Gene conversion vs point mutation in generating variability at the antigen recognition site of major histocompatibility complex loci

We have prepared a [³²p]-labeled oligonucleotide probe carrying a ureido (-NH-CO-NH₂) function at its 3-terminus. This labeled oligomer was used to study polycondensations of urea and formaldehyde and of various phenols and formaldehyde in aqueous solution. The formation of formaldehyde copolymers attached to the amido-function of the probe was monitored by gel electrophoresis. Our results are generally in agreement with those obtained using conventional techniques. Our method is suitable for monitoring potentially prebiotic polycondensation reactions involving formaldehyde.Abbreviations HBA 4-hydroxybenzoic acid, Na⁺ salt - HBzOH 2-hydroxybenzyl alcohol - HBSA 4-hydroxybenzenesulfonic acid, Na⁺ salt Correspondence to: L.E. Orgel     

Polymerization of coniferyl alcohol by Mn3+‐mediated (enzymatic) oxidation: Effects of H2O2 concentration,aqueous organic solvents,and pH

The objective of this study was to evaluate the ability of one versatile peroxidase and the biocatalytically generated complex Mn(III)‐malonate to polymerize coniferyl alcohol (CA) to obtain dehydrogenation polymers (DHPs) and to characterize how closely the structures of the formed DHPs resemble native lignin. Hydrogen peroxide was used as oxidant and Mn²⁺ as mediator. Based on the yields of the polymerized product, it was concluded that the enzymatic reaction should be performed in aqueous solution without organic solvents at 4.5 ≤ pH ≤ 6.0 and with 0.75 ≤ H₂O₂:CA ratio ≤ 1. The results obtained from the Mn³⁺‐malonate‐mediated polymerization showed that the yield was almost 100%. Reaction conditions had, however, effect on the structures of the formed DHPs, as detected by size exclusion chromatography and pyrolysis‐GC/MS. It can be concluded that from the structural point of view, the optimal pH for DHP formation using the presently studied system was 3 or 4.5. Low H₂O₂/CA ratio was beneficial to avoid oxidative side reactions. However, the high frequency of β–β linkages in all cases points to dimer formation between monomeric CA rather than endwise polymerization. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:81–90, 2018     

Effect of Reaction Conditions on the Characterization of Plasmon-Driven Surface Catalytic Reduction Reaction for Para-nitroaniline in a Liquid Condition

Plasmon-driven–coupling reactions through nitro reduction or amino oxidation have been widely reported in the literature. This article studies the functional groups, reaction environment, and the effect of the molecular probe concentration on the coupling reaction using a surface-enhanced Raman spectroscopy (SERS) detection technique. First, we illustrate a sequence of nitro reduction and amino oxidation reactions under the same reaction conditions. Secondly, the relationships between the characterization phenomenon and the variables were studied by adjusting both the pH of the solution, the laser exposure time, and the concentration of the molecular probe in the reaction. We also performed the SERS detection on the product solution in liquid environments, indicating that p,p′-dimercaptoazobenzene (DAAB) is stable in the reaction system. There was no reverse reaction present, and no further polymerization was performed. This study demonstrates that the chemical reaction process is stable in plasmon-catalyzed reactions and serves as a starting point for future fluorescent probe studies with pH-regulated reactions.

     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

Tuning micelles of a bioactive heptapeptide biosurfactant via extrinsically induced conformational transition of surfactin assembly

We have studied the effects of extrinsic environmental conditions on the conformation of surfactin, a heptapeptide biosurfactant from Bacillus subtilis, in aqueous solutions. It has been made clear that temperature, pH, Ca²⁺ ions and the synthetic nonionic surfactant hepta-ethylene glycol (C₁₂E₇) affect the conformation of surfactin in aqueous solutions. The β-sheet formation reached a maximum at 40°C both in presence and absence of (C₁₂E₇) and the nonionic surfactant enhances the β-sheet formation even at 25°C. Ca²⁺ induced the formation of a-helices and caused this transition at 0.3 mm with surfactin monomers or at 0.5 mm with surfactin micelles, but above these transition concentrations of Ca²⁺ β-sheets were observed. In micellar solution the β-sheet structure was stabilized at pH values below 7 or upon addition of Ca²⁺ in concentrations above 0.5 mm . Our results indicated that the bioactive conformation of surfactin is most likely the β-sheets when the molecules are assembled in micelles. The β-sheet structure in micelles could be retained by tuning the micelles. Surfactin micelles could be tuned in the bioactive conformation by manipulating pH, temperature, Ca²⁺ or (C₁₂E₇) concentrations in surfactin solutions. Our results strongly indicated that Ca²⁺ and other molecules (such as C₁₂E₇) may function as directing templates in the assembly and conformation of surfactin in micelles. Thus, we suggest environmental manipulation and template-aided micellation (TAM) as a new approach for preparing predesigned micelles, microemulsions or micro-spheres for specific application purposes. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction

Summary The parameter Tm^t has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tm^t) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tm^t is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tm^t is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.     

A turn‐on luminescence probe for highly selective detection of an anthrax biomarker

Highly selective detection of Bacillus anthrax spores has attracted worldwide attention because Bacillus anthrax spores not only are harmful to the health of human beings and animals, but also can be used as biological warfare agents. Here, we report a simple platform by mixing EuCl₃·6H₂O and sodium polyacrylate in aqueous solution and further investigate its luminescence response towards Bacillus anthrax biomarker dipicolinic acid (DPA) as a turn‐on luminescence probe. Importantly, our probe has good sensitivity, lower detection limit, excellent selectivity as well as great anti‐interference ability due to the great luminescence enhancement of Eu³⁺. Moreover, a test paper is constructed to realize the purpose of portable detection. These results indicate that our probe is an excellent candidate for sensing DPA.     

An evaluation of the conditions necessary for optimal protein A-gold labelling of capsular antigen in ultrathin methacrylate sections of the bacteriumPasteurella haemolytica

Summary The protein A-gold (PAG) probe is a particulate immunocytochemical probe that is eminently suitable for quantification. In order to obtain critical results from the technique, a specific and reproducible probe is needed. To this end, the concentration of probe, the variation of labelling different sections within a single grid, the effect of washing procedures, the variation of labelling with time and temperature and the effect of different storage conditions on the probe have been investigated using PAG labelling of capsular antigen on ultrathin methacrylate sections of the bacteriumPasteurella haemolytica.The results indicate that in this antigen-antibody system, and using a 20nm probe, optimal results are achieved with 2×10¹² particles/ml, a labelling time of 60min at room temperature and the PAG probe, which will have been stored at 4°C, should be between 1-and 5-weeks-old. The efficiency of the probe is tested by evaluating different primary antibody concentrations, by evaluating cross reactions of the primary antibody and by evaluating the relative amounts of antibody against internal components of the bacterium present in different antisera.     

TASSER_low‐zsc: An approach to improve structure prediction using low z‐score–ranked templates

In a variety of threading methods, often poorly ranked (low z‐score) templates have good alignments. Here, a new method, TASSER_low‐zsc that identifies these low z‐score–ranked templates to improve protein structure prediction accuracy, is described. The approach consists of clustering of threading templates by affinity propagation on the basis of structural similarity (thread_cluster) followed by TASSER modeling, with final models selected by using a TASSER_QA variant. To establish the generality of the approach, templates provided by two threading methods, SP³ and SPARKS², are examined. The SP³ and SPARKS² benchmark datasets consist of 351 and 357 medium/hard proteins (those with moderate to poor quality templates and/or alignments) of length ≤250 residues, respectively. For SP³ medium and hard targets, using thread_cluster, the TM‐scores of the best template improve by ～4 and 9% over the original set (without low z‐score templates) respectively; after TASSER modeling/refinement and ranking, the best model improves by ～7 and 9% over the best model generated with the original template set. Moreover, TASSER_low‐zsc generates 22% (43%) more foldable medium (hard) targets. Similar improvements are observed with low‐ranked templates from SPARKS². The template clustering approach could be applied to other modeling methods that utilize multiple templates to improve structure prediction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Methacrylate polymerization by AzoRNA: potential usefulness for chromosomal localization of genes

An azo pyrimidine nucleotide has been prepared and enzymatically attached to oligo(A) primers. The nucleotide's azo pyrimidine group has previously been shown to initiate polymerization of methacrylate esters designed to bind marker groups for visualization by microscopy. When attached to RNA molecules complementary to a chromosomal DNA segment, these nucleotides may allow localization of the DNA segment following in situ hybridization of the probe, methacrylate polymerization, and marker attachment. Since mRNA molecules of potential interest as probes bear a 3′-poly(A) tail, the modified nucleotides were added to oligo(A) primers as models. First, N⁴-ureidocytosine nucleotides were enzymatically added to ApApA, (Ap)₉A, or [5′-³²P]-(pA)₁₀, using the modified cytidine 5′-diphosphate and “primer-dependent” polynucleotide phosphorylase (M. luteus). In the case of the ApApA-primed reaction, the N⁴-ureidocytosine nucleotides in the product polynucleotide were converted into azo nucleotides by oxidation with N-bromosuccinimide. The other two primers were employed to study the time course of polynucleotide formation and to verify that primer was indeed being utilized by the enzyme. The suitability of the modified nucleotide for in situ hybridization studies was examined. Poly(N⁴-ureidocytidylic acid) was prepared from poly(C) and semicarbazide by the bisulfite-catalyzed transamination reaction. It was found that 95% of the N⁴-ureidocytosine nucleotides in this polynucleotide survive the elevated temperatures typically required for DNA:DNA denaturation and RNA:DNA annealing. When poly(N⁴-ureidocytidylic acid) was mixed with poly(I) in buffered aqueous salt solutions, no evidence for hybridization was found, so binding of the probe RNA to the denatured chromosomal DNA molecule via the modified nucleotides is not expected. Upon oxidation of poly(N⁴-ureidocytidylic acid) with N-bromosuccinimide, the azo nucleotides were formed, as judged by the appearance of a characteristic peak at approximately 350 nm in the uv-absorption spectrum of the yellow-orange product, azoRNA. The azo nucleotides in azoRNA exhibited the expected acid lability, which is known to be accompanied by 1-glyceryl methacrylate polymerization in the case of the simple azo pyrimidine. Because 1-glyceryl metharcylate bears substituent glycol groups for attaching heavy atoms or fluorescent markers, it is possible that probe RNA molecules bearing azo nucleotides may be useful for localizing low-multiplicity genes along eukaryotic chromosomes.     

The aprotic electrochemistry of quinones

Quinones play important roles in biological electron transfer reactions in almost all organisms, with specific roles in many physiological processes and chemotherapy. Quinones participate in two-electron, two-proton reactions in aqueous solution at equilibrium near neutral pH, but protons often lag behind the electron transfers. The relevant reactions in proteins are often sequential one electron redox processes without involving protons. Here we report the aprotic electrochemistry of the two half-couples, Q/Q^.- and Q^.?/Q⁼, of 11 parent quinones and 118 substituted 1,4-benzoquinones, 91 1,4-naphthoquinones, and 107 9,10-anthraquinones. The measured redox potentials are fit quite well with the Hammett para sigma (σ_para) parameter. Occasional exceptions can involve important groups, such as methoxy substituents in ubiquinone and hydroxy substituents in therapeutics. These can generally be explained by reasonable conjectures involving steric clashes and internal hydrogen bonds. We also provide data for 25 other quinones, 2 double quinones and 15 non-quinones, all measured under similar conditions.     

An aqueous fluorescent probe for Hg2+ detection with high selectivity and sensitivity

An aqueous fluorescent probe, 1, was developed for the rapid detection of Hg²⁺ with high sensitivity and excellent selectivity. Upon the addition of Hg²⁺ in pure aqueous media, the Hg²⁺‐mediated hydrolysis of vinyl ether and subsequent cyclization reactions converted probe 1 into the corresponding iminocoumarin dye, which is strongly fluorescent when excited. The application of this probe for the detection of intracellular Hg²⁺ was successfully demonstrated in living cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Biomesogenic Matrix Systems

Abstract

The bifunctionally reactive nucleoside and distant nucleoside analogs adenosine (Ado), S-[(adenine-9-yl)methoxyethyl]-L-cysteine (Na-salt) (cysA) and 9-vinyladenine (vA) in aqueous solutions assemble on complementary polyuridylic acid templates to form complex lyomesophases. The systems are investigated by polarizing microscopy, differential scanning calorimetry (DSC) and ¹H- and ³¹P-nmr spectroscopies, assisted by molecular modeling studies. The results indicate the importance of biomesogenic (pre)ordering in nucleic acid native and artificial matrix reactions.     

Rapid measurement of drug release from temperature-sensitive liposomes by electron paramagnetic resonance and radioisotope techniques

We have developed two new methods for quantifying drug release from temperature-sensitive liposomes. Large unilamellar vesicles were made by the reverse phase evaporation process. They contained a water-soluble electron paramagnetic resonance probe, trimethyl-4-amino-2,2,6,6-tetramethyl piperidine N-oxyl and the radioisotope cytosine-[³H]1-β-D-arabinofuranoside in their aqueous compartment. Release of the electron paramagnetic resonance probe was measured by placing the liposomes in a solution of a spin label quenching agent, potassium ferricyanide, and monitoring the reduction in signal strength. The measurement of radioisotope release involved rapid ultracentrifugation of the liposomes after which the supernatant was tested for the presence of radioactivity. Both methods were found to be rapid and convenient ways of measuring drug release from temperature-sensitive liposomes and both methods gave comparable results. The radioisotope assay provides a direct measurement of drug leakage, whereas the electron spin resonance assay provides a continuous marker for liposome stability as a function of temperature.     

Synthesis and characterization of a novel MPEG–chitosan diblock copolymer and self-assembly of nanoparticles

In this paper, a simple and novel method based on free-radical polymerization initiated by potassium persulfate (KPS) was developed to synthesize the MPEG–chitosan diblock copolymer (MPEG–CS). The obtained MPEG–CS diblock copolymer was characterized by Fourier transform infrared (FTIR), ¹H nuclear magnetic resonance (¹H NMR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC), respectively. The MPEG–CS copolymer could self-assemble into nanoparticles in aqueous solution. A typical TEM photography indicated that the well-spherical nanoparticles with diameter at about 200 nm were obtained. In vitro cell culture assay indicated that MPEG–CS nanoparticles are non-toxic and cell-compatible as the polymer concentration was smaller than 0.6 mg/ml. In conclusion, the obtained MPEG–CS nanoparticles might have great potential application in drug-delivery system.     

Structured Functional Domains of Myelin Basic Protein: Cross Talk between Actin Polymerization and Ca-Dependent Calmodulin Interaction

The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca²⁺-activated calmodulin (Ca²⁺-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22–K56), (S72–S107), and (S133–S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca²⁺-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca²⁺-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca²⁺-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca²⁺-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.     

A 59Co NMR relaxation probe of macromolecule anionic binding sites

⁵⁹Co NMR linewidth measurements are reported which show that hexacyanocobaltate-(III) ion may be used as a sensitive probe of protein interactions with anions in aqueous solutions. Applications are demonstrated to bovine serum albumin where the probe ion binding is monitored as a function of pH and is displaced from the protein sites by hexacyanoferrate(III) ion. The general utility of complex metal ions is suggested as a generally useful approach to the analysis of ion-macromolecule interactions.     

Degradation kinetics and mechanism of RH1, a new anti-tumor agent: A technical note

Summary and Conclusions  The degradation of RH1 in aqueous solution is found to be both acid and base catalyzed. The maximum stability is obtained in neutral pH but still degrades by 10% (t₉₀) after just 1 week. The stability profile at pH 5 was done, and 4 major degradation products were observed in acid solutions. LC-MS was performed and the molecular weights determined, from which a degradation mechanism was proposed. Degradation products I, II, and III form 2 isomers each depending on which aziridine group is hydrolyzed. No significant effect of light or the presence of antioxidants was observed, indicating that photodegradation and oxidation are not likely degradation reactions. Published: March 2, 2007     

Kinetics of biopolymerization on nucleic acid templates

The kinetics of biopolymerization on nucleic acid templates is discussed. The model introduced allows for the simultaneous synthesis of several chains, of a given type, on a common template, e.g., the polyribosome situation. Each growth center [growing chain end plus enzyme(s)] moves one template site at a time, but blocks L adjacent sites. Solutions are found for the probability n_j(t) that a template has a growing center that occupies the sites j — L + 1,…, j at time t. Two special sets of solutions are considered, the uniform-density solutions, for which n_j(t) = n, and the more general steady-state solutions, for which dn_j(t)/dt = 0. In the uniform-density case, there is an upper bound to the range of rates of polymerization that can occur. Corresponding to this maximum rate, there is one uniform solution. For a polymerization rate less than this maximum, there are two uniform solutions that give the same rate. In the steady-state case, only L = 1 is discussed. For a steady-state polymerization rate less than the maximum uniform-density rate, the steady-state solutions consist of either one or two regions of nearly uniform density, with the density value(s) assumed in the uniform region(s) being either or both of the uniform-density solutions corresponding to that polymerization rate. For a steady-state polymerization rate equal to or slightly larger than the maximum uniform-density rate, the steady-state solutions are nearly uniform to the single uniform-density solution for the maximum rate. The boundary conditions (rate of initiation and rate, of release of completed chains from the template) govern the choice among the possible solutions, i.e., determine the region(s) of uniformity and the value(s) assumed in the uniform region(s).     

Studies on the Degradation Mechanisms of Proteins and their Derivatives in the Foods

Cysteine-aldehyde compounds were prepared by the reactions of l-cysteine with formaldehyde, acetaldehyde, n-butyraldehyde, benzaldehyde and furfural in 50% ethanol solutions. Hydrogen sulfide and ammonia liberated from cysteine-aldehyde compounds in heated aqueous solutions (oil bath : 120°C) were determined. Although thiazolidine derivatives were stable generally in boiling aqueous solution, l-cysteine-furfural compound was unstable and a large amount of hydrogen sulfide compared with other compounds was released.