Asparagine-135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket

This work studies the structure-function relationships of Asn¹³⁵, a residue situated in the GTP binding pocket of elongation factor Tu (EF-Tu). For this purpose we constructed EF-TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF-TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn¹³⁵ side-chain does not recognize the exocyclic keto group of the guanine base. However, EF-TuN 135D/D 138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa-tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn¹³⁵ is important for the correct folding of the nucleotide binding site and that EF-Tu·kirromycin can mediate the binding of aa-tRNA to the mRNA-programmed ribosomes independently of the native conformation of this site.     

Roosting Requirements of Two Frugivorous Bats (Sturnira lilium and Arbiteus intermedius) in Fragmented Neotropical Forest1

As tropical forest fragmentation accelerates, scientists are concerned with the loss of species, particularly those that play important ecological roles. Because bats play a vital role as the primary seed dispersers in cleared areas, maintaining healthy bat populations is critical to natural forest regeneration. Observations of foraging bats suggest that many Neotropical fruit‐eating species have fairly general habitat requirements and can forage in many different kinds of disturbed vegetation; however, their roosting requirements may be quite different. To test whether or not general foraging requirements are matched by equally broad roosting requirements, we used radiotelemetry to locate roost sites of two common frugivorous bat species (Sturnira lilium and Artibeus intermedius) in a fragmented forest in southeastern Mexico. Sturnira lilium roosted inside tree cavities and selected large‐diameter roost trees in remnant patches of mature forest. Fewer than 2 percent of trees surveyed had a mean diameter equal to or greater than roost trees used by . S. lilium, Artibeus intermedius roosted externally on branches and vines and under palm leaves and selected roost trees of much smaller diameter. Compared to random trees, roost trees chosen by A. intermedius were closer to neighboring taller trees and also closer in height to these trees. Such trees likely provide cryptic roosts beneath multiple overlapping crowns, with sufficient shelter from predators and the elements. While males of A. intermedius generally roosted alone in small trees within secondary forest, females roosted in small groups in larger trees within mature forest and commuted more than three times farther than males to reach their roost sites. Loss of mature forest could impair the ability of frugivorous bats to locate suitable roost sites. This could have a negative impact on bat populations, which in turn could decrease forest regeneration in impacted areas.     

Trypanosoma brucei: Maintenance of concentrated suspensions of bloodstream trypomastigotes in vitro using continuous dialysis for measurement of endocytosis

A continuous dialysis technique capable of maintaining concentrated suspensions of bloodstream Trypanosoma brucei (up to 4 × 10⁸/ml) at 25 C for 2 hr without loss of viability has been developed in order to measure endocytosis under controlled conditions in vitro. Using this technique, the kinetics and mechanism of uptake of the metabolically inert macromolecule, ¹²⁵I-labelled polyvinylpyrrolidone (¹²⁵I-PVP), have been investigated. Binding to the plasma membrane and the rate of uptake of ¹²⁵I-PVP from the extracellular medium by the trypanosome are both decreased by the addition of unlabelled PVP and human serum albumin. A mechanism for uptake of ¹²⁵I-PVP by a combination of fluid-phase and adsorptive endocytosis from the flagellar pocket of the trypanosome is proposed. In the presence of serum albumin and unlabelled PVP, endocytosis of ¹²⁵I-PVP occurs in the fluid phase only, with endocytic indices of 14.5 ± 0.9 and 54.1 ± 11.3 nl/hr/mg protein in vitro at 25 C and in vivo at 37 C, respectively.     

Ultrastructural studies on the secretory cavities ofCitrus deliciosa ten. I. Early stages of the gland cells differentiation

Summary Secretory cavities ofCitrus deliciosa seem to originate from a pair of meristematic cells (an epidermal cell and a second one placed under it). These cells undergo successive divisions resulting in the formation of a globular/oval gland situated in the parenchyma, and a conical stalk, which joins the gland with the epidermis. The young gland consists of a central group of polyhedral cells ensheathed by layers of radially flattened cells.During the early differentiation stages of the gland cells a close association of cytoplasmic microtubules with various organelles is observed. Plastids increase progressively in number and size and their matrix locally contains tubular networks accompanied by small oil droplets. In peripheral cytoplasm numerous myelin-like lomasomes have been observed. Peripheral cells of the gland are gradually modified from the inner cells following a different developmental pattern. Their walls become thicker and plastids do not contain tubular complexes, but only a few thylakoids partly surrounding the newly formed starch grains.     

肝素C5异构酶的表达优化及分子改造

肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase,C5,EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶,催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5号位羧基翻转生成L-艾杜糖醛酸 (L-iduronic acid,L-IdoA)。文中以大肠杆菌Escherichia coli为宿主对斑马鱼来源的肝素葡萄糖醛酸C5异构酶基因Glce进行重组表达优化与分子改造。比较了3种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5表达的差异情况,其中以嗜冷启动型载体pCold Ⅲ表达酶活最高,达到(1 873.61±5.42) U/L。为了进一步提高C5的可溶表达量,在N端融合促溶标签SET2后,可溶蛋白表达量比对照提高了50%,酶活达到 (2 409.25±6.43) U/L。在此基础上,通过理性设计对底物结合口袋进行定点突变,获得最优突变体 (V153R) 的酶活和比酶活分别为 (5 804.32±5.63) U/L和(145.14±2.33) U/mg,是原始酶的2.41倍和2.28倍。肝素C5异构酶改造与表达优化为酶法催化合成肝素奠定了基础。     

Structural,energetic, and dynamic responses of the native state ensemble of staphylococcal nuclease to cavity‐creating mutations

The effects of cavity‐creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C‐terminal domain and the interface between α and β subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the β‐barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions. © 2012 Wiley Periodicals, Inc.     

Acidic biphenyl derivatives: Synthesis and biological activity of a new series of potent 5-HT4 receptor antagonists

Serotonin (5-hydroxytryptamine, 5-HT) is an important signaling molecule in the central nervous system (CNS) and in non-neuronal tissues and organs. Serotonin mediates a positive chronotropic and inotropic response through 5-HT₄ receptors in the atrium and ventricle of the heart. Recent investigations have revealed increased expression of the 5-HT_4(b) isoform in cardiomyocytes of chronic arrhythmic and failing hearts, and that the use of 5-HT₄ receptor antagonists may be beneficial for treating these conditions. The 5-HT₄ receptor possesses a transmembrane (TM) binding site important for ligand affinity and recognition, as well as a capacity to accommodate bulky ligands. A new series of peripherally-acting 5-HT₄ receptor antagonists were prepared by combining the acidic biphenyl group from the class of angiotensin II receptor blockers (ARBs) with the SB207266 (piboserod) scaffold. The new compounds were pharmacologically evaluated and carboxylic acid 21 was identified as a potent and promising 5-HT₄ receptor antagonist with moderate affinity for the AT₁ receptor. The permeability of carboxylic acid 21 in a Caco-2 assay was low and the corresponding prodrug esters 23a–f were therefore prepared. The pharmacokinetics of methyl ester 20 and n-butyl ester 23c were evaluated in a rat model, revealing incomplete metabolism to carboxylic acid 21. However, methyl ester 20 is a potent 5-HT₄ receptor antagonist with binding affinities in the low picomolar range. Methyl ester 20 has promising oral bioavailability and pharmacokinetics and may target 5-HT₄ receptors in both CNS and peripheral organs.     

On the Permeation by Dioxygen of Urate Oxidase from Aspergillus flavus in Complex with Xanthine Anion: Dioxygen Pathways and a Portrait of the Enzyme Cavities from Molecular Dynamics Simulations in Water Solution

This work describes molecular dynamics (MD) simulations in aqueous media for the complex of the homotetrameric urate oxidase (UOX) from Aspergillus flavus with xanthine anion ( 5 ) in the presence of dioxygen (O₂). After 196.6 ns of trajectory from unrestrained MD, a O₂ molecule was observed leaving the bulk solvent to penetrate the enzyme between two subunits, A/C. From here, the same O₂ molecule was observed migrating, across subunit C, to the hydrophobic cavity that shares residue V227 with the active site. The latter was finally attained, after 378.3 ns of trajectory, with O₂ at a bonding distance from 5 . The reverse same O₂ pathway, from 5 to the bulk solvent, was observed as preferred pathway under random acceleration MD (RAMD), where an external, randomly oriented force was acting on O₂. Both MD and RAMD simulations revealed several cavities populated by O₂ during its migration from the bulk solvent to the active site or backwards. Paying attention to the last hydrophobic cavity that apparently serves as O₂ reservoir for the active site, it was noticed that its volume undergoes ample fluctuations during the MD simulation, as expected from the thermal motion of a flexible protein, independently from the particular subunit and no matter whether the cavity is filled or not by O₂.     

A mast‐seeding desert shrub regulates population dynamics and behavior of its heteromyid dispersers

Granivorous rodent populations in deserts are primarily regulated through precipitation‐driven resource pulses rather than pulses associated with mast‐seeding, a pattern more common in mesic habitats. We studied heteromyid responses to mast‐seeding in the desert shrub blackbrush (Coleogyne ramosissima), a regionally dominant species in the Mojave–Great Basin Desert transition zone. In a 5‐year study at Arches National Park, Utah, USA, we quantified spatiotemporal variation in seed resources in mast and intermast years in blackbrush‐dominated and mixed desert vegetation and measured responses of Dipodomys ordii (Ord's kangaroo rat) and Perognathus flavescens (plains pocket mouse). In blackbrush‐dominated vegetation, blackbrush seeds comprised >79% of seed production in a mast year, but 0% in the first postmast year. Kangaroo rat abundance in blackbrush‐dominated vegetation was highest in the mast year, declined sharply at the end of the first postmast summer, and then remained at low levels for 3 years. Pocket mouse abundance was not as strongly associated with blackbrush seed production. In mixed desert vegetation, kangaroo rat abundance was higher and more uniform through time. Kangaroo rats excluded the smaller pocket mice from resource‐rich patches including a pipeline disturbance and also moved their home range centers closer to this disturbance in a year of low blackbrush seed production. Home range size for kangaroo rats was unrelated to seed resource density in the mast year, but resource‐poor home ranges were larger (P < 0.001) in the first postmast year, when resources were limiting. Blackbrush seeds are higher in protein and fat but lower in carbohydrates than the more highly preferred seeds of Indian ricegrass (Achnatherum hymenoides) and have similar energy value per unit of handling time. Kangaroo rats cached seeds of these two species in similar spatial configurations, implying that they were equally valued as stored food resources. Blackbrush mast is a key resource regulating populations of kangaroo rats in this ecosystem.     

A molecular docking study of estrogenically active compounds with 1,2-diarylethane and 1,2-diarylethene pharmacophores

Numerous selective estrogen receptor modulators (SERMs) have been synthesized and assayed in recent years. The focus of this study is to apply coarse-grain molecular docking procedures coupled with fine-grain all-atom force field optimization strategies to shed light on the binding mechanisms of currently available estrogen receptor-active compounds. Although the mechanics of ligand binding in estrogen receptors is generally well understood, there is room for surprises. In this paper computational evidence corroborating the experimentally observed type I agonistic binding mode for estradiol (E2) and diethylstilbesterol (DES) and the type II antagonistic binding mode for 4-hydroxytamoxifen and raloxifen is presented. Included in this type I agonistic mode are the DES derivatives, transstilbene and 1,2-diaryldiaminoethane. In addition, a novel ‘type II agonistic’ binding mode for 2,3-diarylimidazolines, 4,5-diarylimidazoles, 2,3-diarylpiperazines is introduced. This mode is stabilized by suggesting alternative hydrogen bond anchor points in the ligand binding domain as potential leads for future drug design.