Stabilization of a tetrameric enzyme (α-amino acid ester hydrolase from Acetobacter turbidans) enables a very improved performance of ampicillin synthesis

The stabilized derivative of the enzyme α-amino acid ester hydrolase from Acetobacter turbidans has been found to be very adequate as biocatalyst of the synthesis of the very relevant antibiotic ampicillin. This enzyme resulted much more adequate than the Penicillin G Acylase (PGA) from Escherichia coli (the most used enzyme). The stabilization of the enzyme was required because under optimal conditions (absence of phosphate and 40% of MeOH), no-stabilized derivatives or soluble enzyme from A. turbidans become very rapidly inactivated. Under these conditions, this new stabilized derivative exhibited a very high selectivity for the transferase activity compared to the esterase one, as well as a very low hydrolytic activity towards the antibiotic. Moreover, this new biocatalyst did not recognize -phenylglycine as substrate in the synthetic process. By using the racemic mixture of / phenylglycine methyl ester, 85% of the -ester could be transformed to ampicillin. In contrast, the enzyme from E. coli exhibited a high hydrolytic activity for the ampicillin yielding low synthetic yields. This enzyme also resulted much less enantioselective producing both isomers of the antibiotic.     

Dimerization of CtIP may stabilize in vivo interactions with the Retinoblastoma-pocket domain

CtIP is a tumor suppressor that interacts with Retinoblastoma protein (Rb) to regulate the G1/S-phase transition of the cell cycle. Despite its large size (897 residues) CtIP has few known structured regions. Rather it contains several linear motifs that interact with known binding partners, including an LXCXE motif that binds the pocket domain of Rb-family proteins. This LXCXE motif lies at the C-terminus of the only known structured domain, an N-terminal coiled-coil dimerization domain (DD; residues 45-160). Yeast two-hybrid (Y2H) and GST-pulldown analyses showed that CtIP requires the LXCXE motif to bind the Rb-pocket. Although isothermal titration calorimetry data indicates that the LXCXE motif is the sole determinant of binding affinity for the Rb-pocket domain (K(A) approximately 10(6)M(-1)), Y2H data indicates that the DD is required to stabilize the interaction in vivo. Thus dimerization may increase the apparent stability of the proteins and/or the lifetime of the complexes.     

Synthesis, spectroscopic, structural and electrochemical studies of carboxyl substituted 1,4-naphthoquinones

Two carboxyl substituted quinones and their ethyl esters were prepared by alkylation of 2-methyl-1,4-naphthoquinone (MNQ), also known as menadione or vitamin K₃. All products were characterized by spectroscopic (¹H NMR, ¹³C NMR, IR) and electrochemical (cyclic voltammetry) methods, and the crystal structure of the two carboxylic derivatives was also determined. Both carboxyl substituted quinones crystallize in the system as hydrogen bonded dimers. In MeCN, the cyclic voltammograms of the ester derivatives present two reversible one-electron redox waves very similar to those of the parent quinone, MNQ. However, in the same solvent, the corresponding carboxyl substituted quinones show one cathodic and one anodic additional irreversible waves at more positive potentials and a decrease in current intensity of the two quinone reduction waves accompanied by loss of the quasi-reversible character of the second wave. These results show that the presence of the carboxylic substituent does not greatly modify the redox behaviour of the quinone, except for a small anodic shift of the potentials, but the associated presence of H⁺ ions in solution causes an important perturbation to the system, stabilizing the electrogenerated semiquinones by intermolecular self-protonation and/or hydrogen bonding.     

The role of the cytoskeleton in the formation of gap junctions by Connexin 30

Mutations in the genes that encode Connexin 26 (GJB2) and Connexin 30 (GJB6) are the most common known cause of hereditary nonsyndromic sensorineural deafness. Cx26 and Cx30 share a similar protein structure, as well as the same expression distribution pattern in the cochlea. Cx26 has different intracellular trafficking properties compared to those of Cx43 and Cx32, whose trafficking manner is consistent with the classical membrane protein secretory pathway. Until now, however, the trafficking patterns of Cx30 have not been studied. By means of an immunofluorescence staining approach, we found that the targeting of Cx30 to gap junctions in transfected HeLa cells is not affected by brefeldin A, suggesting a Golgi-independent feature, similar to Cx26. Nocodazole had a minimal effect on assembly and distribution of Cx30 gap junctions. Cytochalasin B-induced actin filament depolymerization, however, affected both the pattern and the distribution of Cx30 gap junctions. Co-localization with and/or interaction between Cx30 and microtubules and cortical actin filaments, but not with the tight/adherens junction protein ZO-1, was confirmed by immunofluorescence and/or immunoprecipitation methods. The results suggest that the cytoskeleton, and especially actin filaments, are important components in the processes of assembly, trafficking and stabilization of Cx30 gap junctions.     

Converting the highly amyloidogenic human calcitonin into a powerful fibril inhibitor by three-dimensional structure homology with a non-amyloidogenic analogue

Irreversible aggregation limits bioavailability and therapeutic activity of protein-based drugs. Here we show that an aggregation-resistant mutant can be engineered by structural homology with a non-amyloidogenic analogue and that the aggregation-resistant variant may act as an inhibitor. This strategy has successfully been applied to the amyloidogenic human calcitonin (hCT). Including only five residues from the non-amyloidogenic salmon calcitonin (sCT), we obtained a variant, polar human calcitonin (phCT), whose solution structure was shown by CD, NMR, and calculations to be practically identical to that of sCT. phCT was also observed to be a potent amyloidogenesis inhibitor of hCT when mixed with it in a 1:1 ratio. Fibrillation studies of phCT and the phCT-hCT mixture mimicked the sCT behavior in the kinetics and shapes of the fibrils with a dramatic reduction with respect to hCT. Finally, the effect of phCT alone and of the mixture on the intracellular cAMP level in T47D cells confirmed for the mutant and the mixture their calcitonin-like activity, exhibiting stimulation effects identical to those of sCT, the current therapeutic form. The strategy followed appears to be suitable to develop new forms of hCT with a striking reduction of aggregation and improved activity. Finally, the inhibitory properties of the aggregation-resistant analogue, if confirmed for other amyloidogenic peptides, may favor a new strategy for controlling fibril formation in a variety of human diseases.     

A stabilizing and denaturing dual-effect for natural polyamines interacting with G-quadruplexes depending on concentration

Both G-quadruplexes and natural polyamines are intimately associated with tumor growth and proliferation. The effect of the natural polyamines on telomeric and some oncogenic G-quadruplexes including bcl-2, c-kit, and c-myc G-quadruplexes has been studied by using absorption, fluorescence, CD, and NMR methods. The results exhibited an interesting dual-effect depending on polyamine? concentration. Polyamines promote and stabilize G-quadruplexes under a lower concentration (less than 1 mM) but denature G-quadruplexes under a higher concentration (more than 1 mM). Probably the electrostatic and hydrophobic effect of polyamines and the hydrogen-bonding interaction between guanines and polyamines were respectively responsible for the stabilizing and denaturing effect.     

Endcaps for Stabilizing Short DNA Duplexes

Abstract

The syntheses of endcaps for covalently linking the 3′ and 5′ hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by T_m experiments on the self-complementary octamer.     

On the stabilization of animal numbers. Problems of testing

Summary When testing census data of insect populations for regulation, and/or for overall density dependence in the course of numbers over years, certain conditions, which follow from the testing models, should be fulfilled. Even if the series of densities may be considered a piece of first-order Markov chain (a necessary condition) significant test results need not obviously point to regulation of numbers by dominant density-dependent processes. Such a case is presented by the pine looper population at Hoge Veluwe studied by Klomp. A drastic drop in density from 1952 to 1953, which takes 78–97% of the log-density range (LR) over all years, most probably wrongly causes significant test results. This is supported by some simulation experiments. Moreover, we cannot be sure that the population was sufficiently isolated, i.e. that dispersal of adults from surrounding populations did not importantly influence population numbers. Among 6 Panolis-populations studied by Schwerdtfeger during 17 years a single one scored significantly with all tests. This resulted, however, from such a drastic drop in density that it covered the entire log-density range (LR=9.39), which therefore is wider than in any of the other (non-significant) populations. Another Panolis-population that maintained itself during 60 years, and which also scored significantly, most probably was kept within limits by supplementation of very low densities with immigrants, on the one hand, and by restriction of high densities by defoliation caused by other species, on the other. It is discussed whether this can be considered regulation, or results from spreading of risk. It is concluded that the range stability of particular populations must be considered generally to be the result of stabilization by both internal and external processes among which both density-dependent and density-independent processes play a significant part, and from which the contribution of the density-dependent processes need not be separated. The most interesting aspect of the stabilization of animal numbers is its relationship with the expected survival time of the population.Communication No. 402 of the Biological Station, Wijster     

Biological synthesis of nanoparticles in biofilms

The biological synthesis of nanoparticles (NPs) by bacteria and biofilms via extracellular redox reactions has received attention because of the minimization of harmful chemicals, low cost, and ease of culturing and downstream processing. Bioreduction mechanisms vary across bacteria and growth conditions, which leads to various sizes and shapes of biosynthesized NPs. NP synthesis in biofilms offers additional advantages, such as higher biomass concentrations and larger surface areas, which can lead to more efficient and scalable biosynthesis. Although biofilms have been used to produce NPs, the mechanistic details of NP formation are not well understood. In this review, we identify three critical areas of research and development needed to advance our understanding of NP production by biofilms: 1) synthesis, 2) mechanism and 3) stabilization. Advancement in these areas could result in the biosynthesis of NPs that are suitable for practical applications, especially in drug delivery and biocatalysis. Specifically, the current status of methods and mechanisms of nanoparticle synthesis and surface stabilization using planktonic bacteria and biofilms is discussed. We conclude that the use of biofilms to synthesize and stabilize NPs is underappreciated and could provide a new direction in biofilm-based NP production.     

硒对红细胞膜稳定作用的浓度依赖性

 Na_2SeO_3对人红细胞膜骨架具有稳定作用,但这种作用依赖于Na_2SeO_3的浓度。在低离子强度下,4℃透析人红细胞膜,实验组加入不同浓度的Na_2SeO_3,对照组不加Na_2SeO_3。结果表明,0.1—0.8ppm Na_2SeO_3的存在比对照组具有较高的Na~+,K~+-ATP酶活性、膜脂流动性。用N-[3-芘]-马来酰胺作探针,反映两者构象也有差异。如果在透析液中加入较高浓度的Na_2SeO_3(>1.0ppm)则会产生与低浓度相反的结果。人红细胞膜~31P-NMR的测试也表明,加入0.4ppm与4.0ppm Na_2SeO_3会产生不同的结果。与对照组相比较,低浓度使化学位移各向异性值(△σ)下降,而高浓度则使△σ增加。