Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

Background  

Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.     

Focal reduction of αE-catenin causes premature differentiation and reduction of β-catenin signaling during cortical development

Cerebral cortical precursor cells reside in a neuroepithelial cell layer that regulates their proliferation and differentiation. Global disruptions in epithelial architecture induced by loss of the adherens junction component αE-catenin lead to hyperproliferation. Here we show that cell autonomous reduction of αE-catenin in the background of normal precursors in vivo causes cells to prematurely exit the cell cycle, differentiate into neurons, and migrate to the cortical plate, while normal neighboring precursors are unaffected. Mechanistically, αE-catenin likely regulates cortical precursor differentiation by maintaining β-catenin signaling, as reduction of αE-catenin leads to reduction of β-catenin signaling in vivo. These results demonstrate that, at the cellular level, αE-catenin serves to maintain precursors in the proliferative ventricular zone, and suggest an unexpected function for αE-catenin in preserving β-catenin signaling during cortical development.     

DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical–biological flocculation and chemical coagulation systems

Microbial community DNA was extracted from activated sludge samples taken from a chemical bioflocculation process and a chemical coagulation process in Shanghai, China. 16S rDNA of ammonia-oxidizing bacteria (AOB)was amplified by nested polymerase chain reaction and fingerprinted by denaturing gradient gel electrophoresis for microbial structure analysis. The Shannon diversity index of each sample was determined. The results indicated that the microbial structure of AOB in chemical bioflocculation process was comparable at two operational conditions. The ammonia-oxidizing bacterial communities were similar in three channels of the chemical bioflocculation process and in three serial tanks in the chemical coagulation process at the same condition. The diversity of microbial structures in the chemical bioflocculation process was higher than in the chemical coagulation process, in which the microbial structure was similar to that in the influent. Although the microbial study provides insights to the nitrification removal, higher microbial diversity of AOB does not necessarily mean higher ammonia oxidization. Molecular analysis should be combined with chemical assays to optimize operational conditions.     

Deficient repair of chemical adducts in α DNA of monkey cells

We have examined excision repair of DNA damage in the highly repeated α DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in α DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for α DNA and bulk DNA. In cells treated with furocoumarins and long-wavelength ultraviolet light, however, repair synthesis in α DNA was only 30% of that in bulk DNA, although it followed the same time course. We found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as we found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from α DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in α DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences.     

Combined chemical shift changes and amino acid specific chemical shift mapping of protein–protein interactions

Protein–protein interactions are often studied by chemical shift mapping using solution NMR spectroscopy. When heteronuclear data are available the interaction interface is usually predicted by combining the chemical shift changes of different nuclei to a single quantity, the combined chemical shift perturbation In this paper different procedures (published and non-published) to calculate are examined that include a variety of different functional forms and weighting factors for each nucleus. The predictive power of all shift mapping methods depends on the magnitude of the overlap of the chemical shift distributions of interacting and non-interacting residues and the cut-off criterion used. In general, the quality of the prediction on the basis of chemical shift changes alone is rather unsatisfactory but the combination of chemical shift changes on the basis of the Hamming or the Euclidian distance can improve the result. The corrected standard deviation to zero of the combined chemical shift changes can provide a reasonable cut-off criterion. As we show combined chemical shifts can also be applied for a more reliable quantitative evaluation of titration data.     

Microbial production of chiral pantothenonitrile through stereospecific reduction of 2′-ketopantothenonitrile

Summary A novel enzymatic process for the synthesis of chiral pantothenonitrile through the asymmetric reduction of 2-ketopantothenonitrile is described.Sporidiobolus salmonicolor AKU 4440 was found to convert 2-ketopantothenonitrile (50 mg/ml) to D-(+)-pantothenonitrile (93.6% e.e.), with a molar yield of 95.6%. In a similar manner, conversion to L-(–)-pantothenonitrile occurred on incubation with washed cells ofRhodotorula glutinis AKU 4845 as the catalyst.     

Cholesterol-independent effects of methyl-β-cyclodextrin on chemical synapses

The cholesterol chelating agent, methyl-β-cyclodextrin (MβCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MβCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MβCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MβCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14 °C but not from those acclimatized to 21 °C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MβCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MβCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MβCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MβCD can affect both presynaptic and postsynaptic properties, and that some effects of MβCD are unrelated to cholesterol chelation.     

Microbial production and chemical transformation of poly-γ-glutamate

Poly-γ-glutamate (PGA), a novel polyamide material with industrial applications, possesses a nylon-like backbone, is structurally similar to polyacrylic acid, is biodegradable and is safe for human consumption. PGA is frequently found in the mucilage of natto, a Japanese traditional fermented food. To date, three different types of PGA, namely a homo polymer of d-glutamate (D-PGA), a homo polymer of l-glutamate (L-PGA), and a random copolymer consisting of d- and l-glutamate (DL-PGA), are known. This review will detail the occurrence and physiology of PGA. The proposed reaction mechanism of PGA synthesis including its localization and the structure of the involved enzyme, PGA synthetase, are described. The occurrence of multiple carboxyl residues in PGA likely plays a role in its relative unsuitability for the development of bio-nylon plastics and thus, establishment of an efficient PGA-reforming strategy is of great importance. Aside from the potential applications of PGA proposed to date, a new technique for chemical transformation of PGA is also discussed. Finally, some techniques for PGA and its derivatives in advanced material technology are presented.     

Ab-initio study of anisotropic and chemical surface modifications of β-SiC nanowires

The electronic band structure and electronic density of states of cubic SiC nanowires (SiCNWs) in the directions [001], [111], and [112] were studied by means of Density Functional Theory (DFT) based on the generalized gradient approximation and the supercell technique. The surface dangling bonds were passivated using hydrogen (H) atoms and OH radicals in order to study the effects of this passivation on the electronic states of the SiCNWs. The calculations show a clear dependence of the electronic properties of the SiCNWs on the quantum confinement, orientation, and chemical passivation of the surface. In general, surface passivation with either H or OH radicals removes the dangling bond states from the band gap, and OH saturation appears to produce a smaller band gap than H passivation. An analysis of the atom-resolved density of states showed that there is substantial charge transfer between the Si and O atoms in the OH-terminated case, which reduces the band gap compared to the H-terminated case, in which charge transfer mainly occurs between the Si and C atoms.     

How to tackle chemical communication? Relative proportions versus semiquantitative determination of compounds in lizard chemical secretions

Knowledge about chemical communication in some vertebrates is still relatively limited. Squamates are a glaring example of this, even when recent evidences indicate that scents are involved in social and sexual interactions. In lizards, where our understanding of chemical communication has considerably progressed in the last few years, many questions about chemical interactions remain unanswered. A potential reason for this is the inherent complexity and technical limitations that some methodologies embody when analyzing the compounds used to convey information. We provide here a straightforward procedure to analyze lizard chemical secretions based on gas chromatography coupled to mass spectrometry that uses an internal standard for the semiquantification of compounds. We compare the results of this method with those obtained by the traditional procedure of calculating relative proportions of compounds. For such purpose, we designed two experiments to investigate if these procedures allowed revealing changes in chemical secretions 1) when lizards received previously a vitamin dietary supplementation or 2) when the chemical secretions were exposed to high temperatures. Our results show that the procedure based on relative proportions is useful to describe the overall chemical profile, or changes in it, at population or species levels. On the other hand, the use of the procedure based on semiquantitative determination can be applied when the target of study is the variation in one or more particular compounds of the sample, as it has proved more accurate detecting quantitative variations in the secretions. This method would reveal new aspects produced by, for example, the effects of different physiological and climatic factors that the traditional method does not show.     

Kinetic mechanism of active site assembly and chemical catalysis of DNA polymerase β

It has been inferred from structural and computational studies that the mechanism of DNA polymerases involves subtle but important discrete steps that occur between binding and recognition of the correct dNTP and chemical catalysis. These steps potentially include local conformational changes involving active site residues, reorganization of Mg(2+)-coordinating ligands, and proton transfer. Here we address this broad issue by conducting extensive transient state kinetic analyses of DNA polymerase β (Pol β). We also performed kinetic simulations to evaluate alternative kinetic models. These studies provide some support for two-step subdomain closing and define constraints under which a kinetically significant prechemistry step can occur. To experimentally identify additional microscopic steps, we developed a stopped flow absorbance assay to measure proton formation that occurs during catalysis. These studies provide direct evidence that formation of the enzyme-bound 3'-O(-) nucleophile is rate determining for chemistry. We additionally show that at low pH the chemical step is rate limiting for catalysis, but at high pH, a postchemistry conformational step is rate limiting due to a pH-dependent increase in the rate of nucleotidyl transfer. Finally, we performed exhaustive analyses of [Mg(2+)] and pH effects. In contrast to published studies, the results suggest an irregular pH dependence of k(pol), which is consistent with general base catalysis involving cooperativity between two or more protonic residues. Overall, the results represent significant advancement in the kinetic mechanism of Pol β and also reconcile some computational and experimental findings.     

Heavy metal accumulation of Danube river aquatic plants — indication of chemical contamination

In this paper, the ecological status of a section of the Danube River flowing through Serbia from Bezdan to Djerdap was evalutated. Using the chemical composition of water, sediment samples from the littoral zone and dominant aquatic macrophytes, the level of chemical pollution was ascertained. Chemical analyses of the water and sediment indicated that the tributaries flowing into the Danube significantly influenced the chemical load of the water and as a direct consequence, the sediment. The concentration of heavy metals including Cu, Mn and Cd found in plants of the Potamogeton genus, further indicated significant chemical pollution, establishing a clear link between the chemical composition of plant tissues and the chemical composition of water and sediment. This paper therefore describes how the chemical composition of aquatic plants can be used as a reliable indicator for heavy metal pollution of aquatic ecosystems.     

The influence of storage on the “chemical age” of red wines

Storage conditions and duration have a considerable influence on wine quality. Optimum temperature and humidity conditions may improve wine quality through ageing, while incorrect or excessively long storage leads to negative results. In order to evaluate the global effects of storage on red wine composition, 20 Sangiovese wines were stored in two different conditions (cellar or house) for a period of 2 years and analysed every 6 months. Untargeted LC–MS analysis showed various putative markers for the type and length of conservation (i.e. pigments, flavanols, pantothenic acid etc.), while targeted LC–MS confirmed and expanded these results within specific metabolic groups. The results of multivariate analysis showed that wines stored in the cellar changed little even after 2 years of storage, while wines stored in typical domestic conditions (house) developed approximately four times faster, reaching a composition similar to wines stored in the cellar for 2 years after just 6 months. The formation of several monosulfonated flavanols during domestic ageing provided the first evidence in wine of a reaction between wine tannins—both catechins and proanthocyanidins—and the exogenous antioxidant bisulfite. Moreover, ageing in domestic conditions appeared to induce an accelerated decrease in wine pigments, while specifically promoting the formation of pinotin A-like pigments and the hydrolysis of flavonol glycosides.     

Application of physical–chemical and biological–chemical methods for heavy metals removal from acid mine drainage

Acid mine drainage (AMD) represents a serious environmental problem related to sulfide minerals and coal mining. High content of toxic metals and high acidity in AMD adversely affects surface water, groundwater and soil. The abandoned mine of the Smolník deposit in Slovakia is a typical example in this respect. The quality of AMD needs to be monitored and suitable treatment methods need to be developed.The aim of this paper was to demonstrate the technical feasibility of heavy metals removal from AMD using physical–chemical and biological–chemical methods. The base of the physical–chemical method was electrowinning. The principle of the biological–chemical method was the selective sequential precipitation (SSP) of metals with the application of hydrogen sulfide produced by sulfate-reducing bacteria and sodium hydroxide solution. Both the electrowinning and SSP processes decrease the content of heavy metals in AMD. The pre-treatment of AMD by chemical iron–aluminum precipitation (in the case of electrowinning tests) and chemical iron precipitation (in the case of SSP tests) improved the selectivity of the processes. A further aim of the work was the improvement of the SSP.During the electrochemical experiments, 99% Zn removal – under metallic form – and 94% Mn removal – under MnO₂ form – both with a high degree of purity, were achieved. The SSP process reached the selective precipitation of chosen metals with 99% efficiency – Fe, Al and Mn in the form of metal hydroxides, Cu and Zn as metal sulfides. The results achieved may be used for designing a process appropriate for the selective recovery of metals from the AMD discharged from the Smolník deposit.     

Are chemical barriers necessary for evolution of butterfly-plant associations?

Summary The association between heliconiine butterflies and Passion flower vines is composed of three or more subassociations, in which each Heliconius species group feeds on a different Passiflora subgenus. The relationships are consistent with the adaptive zone hypothesis of Ehrlich and Raven, which would suggest that (1) species of the subgenus Plectostemma proliferated as a result of chemical barriers to herbivory, which created a herbivore-free adaptive zone in which speciation and diversification took place, and (2) species of the H. erato-charitonia group overcame these barriers and entered a competitor-free adaptive zone, in which they proliferated and speciated with those plants as hosts. The hypothesis that plant secondary chemicals were responsible for creating such barriers to herbivory was tested using heliconiine species as bioassays, in which reduced growth rates indicated presence of chemical barriers to feeding. Contrary to expectation, plants of the subgenus Plectostemma showed little or no chemical defense against any species of heliconiine caterpillar. In contrast many plants of the primitive subgenus Granadilla possessed significant chemical barriers against herbivory by heliconiine larvae, excepting those species in the H. numata-melpomene species group. I concluded that chemical barriers to feeding were not responsible for proliferation and diversification in the subgenus Plectostemma, nor did chemicals create a competitor-free adaptive zone in which the H. erato-charitonia species-group could proliferate and speciate. Chemical barriers may have been important in the evolution of the subgenus Granadilla-heliconiine association. I suggest that plant allelochemics are only one of many possible barriers to herbivory which can help create adaptive zones for plants and their herbivores, and that the patterns of butterfly foodplant specialization discussed by Ehrlich and Raven (1964) are not necessarily the result of biochemical adaptation and counteradaptation.     

Use of chemical ionization for GC–MS metabolite profiling

Metabolite profiling is commonly performed by GC–MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e.g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of “unknowns” and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC–MS metabolite profiling. Two reagent gases differing in proton affinity (CH₄ and NH₃) were used to analyse 111 metabolite standards and extracts from plant samples. NH₃-CI mass spectra were simple and generally dominated by [MH]⁺ and/or the adduct [M+NH₄]⁺. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH₃-CI and less than 30% of basepeak in CH₄-CI. With CH₄-CI, [MH]⁺ was generally present but at lower relative abundance than for NH₃-CI. CH₄-CI spectra were commonly dominated by losses of CH₄ [M+1-16]⁺, 1–3 TMSOH [M+1-nx90]⁺, and combinations of CH₄ and TMSOH losses [M+1-nx90-16]⁺. CH₄-CI and NH₃-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH₃-CI and structural information with CH₄-CI.     

Some physical and chemical characteristics of Lake Atnsjøen

The physical and chemical characteristics of the dimictic, ultraoligotrophic, and subalpine Lake Atnsjøen are described based on data from the period 1985–2001. The temperature stratification of the lake is weakly developed, with the thermocline at about 10 m depth. The surface temperature during summer is usually lower than 14–15?°C. The Secchi disk transparency is normally higher than 8–10 m in summer and autumn, but can be as low as 4.3 m during spring. The pH is about 6.0, and the specific conductivity about 1.0 mS m^{? 1}. The dominating cation and anion are Ca (≈0.8 mg l^?1 or 42 μeq l^?1), and SO₄ (≈1.5 mg l^?1 or 33 μeq l^?1), respectively. The concentration of bicarbonate (HCO₃, μeq l^?1) is sometimes lower, sometimes higher than that of SO₄.