Evidence for an Ester Linkage between Lignin and Glucuronic Acid in Lignin–Carbohydrate Complexes by DDQ-Oxidation

The effects of ethanol feeding on the tryptophan-niacin metabolism were investigated in rats. Male rats of the Wistar strain (3 weeks old) were fed with a 20% casein diet and 15% ethanol ad libitum for 56 days. Urine samples were collected every week during the experimental period. Urinary excretion of N¹-methylnicotinamide (MNA) was always higher in the ethanol-fed group than in the control group, but urinary excretion of its oxidized metabolites N¹-methyl-2-pyridone-5-carboxamide (2-Py) and N¹-methyl-4-pyridone-3-carboxamide (4-Py) was always lower. Therefore, the ratio of (2-Py + 4-Py)/MNA excretion was lower in the ethanol-fed group than in the control group. The rats were killed on day 57 and liver enzyme activities involved in the tryptophan-niacin metabolism were also measured. Tryptophan oxygenase, kynureninase, nicotinamide mononucleotide adenylyltransferase, NAD⁺ synthetase, and nicotinamide methyltransferase activities were similar in both groups, but, 2-Py-forming MNA oxidase and 4-Py-forming MNA oxidase activities in the ethanol-fed group were 43% and 18% of the control, respectively. Therefore, the increase in MNA excretion and the decrease in the ratio of (2-Py + 4-Py)/MNA excretion might be attributed to the inhibition of the two MNA oxidase activities by ethanol feeding. Furthermore, it happened to be found that this excretion ratio also increased with growth up to nine weeks and this change was attributed to the increased reaction MNA → 4-Py with growth.     

Analysis of the potential evapotranspiration demands in the Czech Republic between 1961–1990

Dynamics of the evapotranspirational demands in the Czech Republic within three decades from 1961 to 1990 has been studied. The determination of the levels of influence of the respective natural components depends on the regionalized modelling techniques. The project of regionalized modelling is theoretically based on the potential evapotranspiration values (ET ₀) calculated by FAO methodology (eqs. 1, 2) and the series of temperature and rainfall observations obtained by climatologic stations during a 30-year period from 1961 to 1990 and its relation to the absolute altimetric. The DMR-2 military digital elevation model of the Czech Republic relief consisting of a regular network of points with their altitudes specified in meters was used for the purpose of regionalized modelling. One step of the network in the S-42 coordinate system with the Gauss conforms cylindrical projection is equal to 100 meters; this implies that the smallest area for which the data can be processed is 1 hectare. The digital relief model can be linked to both direct (temperature, precipitation) and derived (evapotranspiration) quantities, which is one of the many novelties of regionalized modelling. The climatic data used in regionalized modelling records daily measurements were obtained by 85 climatologic and rainfall-monitoring stations from 1961 to 1990. Our results showed an appreciable decrease of the most drying area (ratio P/ET₀ up to 0.755) in the last decade 1981–90; half as less amounts compared with the previous decade 1971–80 (about 500,000 hectares). On the other hand, an apparent increase (more than 500,000 hectares in comparison with the previous decade 1971–80) of the wettest area (ratio P/ET₀ over 1.508) through the last decade was observed. Both first decades 1961–70 and 1971–80 look similar. The project mentioned in this article has made it possible to create models for the different time intervals which have showed higher reliability for heterogeneous application.     

Analysis of the aplyronine A-induced protein–protein interaction between actin and tubulin by surface plasmon resonance

The antitumor macrolide aplyronine A induces protein–protein interaction (PPI) between actin and tubulin to exert highly potent biological activities. The interactions and binding kinetics of these molecules were analyzed by the surface plasmon resonance with biotinylated aplyronines or tubulin as ligands. Strong binding was observed for tubulin and actin with immobilized aplyronine A. These PPIs were almost completely inhibited by one equivalent of either aplyronine A or C, or mycalolide B. In contrast, a non-competitive actin-depolymerizing agent, latrunculin A, highly accelerated their association. Significant binding was also observed for immobilized tubulin with an actin–aplyronine A complex, and the dissociation constant K_D was 1.84 μM. Our method could be used for the quantitative analysis of the PPIs between two polymerizing proteins stabilized with small agents.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Linkages between common wheat yields and climate in Morocco (1982–2008)

In Morocco, wheat production shows a high inter-annual variability due to uncertain rainfall. In view of the importance of this resource to the country’s economy, it is important to gain a better understanding of the natural large-scale climate oscillation governing this variability. In this study, we analyzed de-trended (1) time series of common wheat yields (1983–2008) from 11 agricultural provinces that account for 80 % of national wheat production; (2) monthly rainfall and 10-day temperature from ten meteorological stations; (3) 10-day normalized difference vegetation index (NDVI) from the AVHRR sensor; (4) monthly atmospheric climate indices [North Atlantic Oscillation (NAO) and Scandinavian Pattern (SCA)] and monthly 500 hPa geopotentials fields; and (5) monthly sea surface temperature (SST) fields and indices (NIÑO3, Tropical North Atlantic and Tropical South Atlantic). The relationship between rainfall and temperature during tillering in early winter and grain filling in early spring and wheat yields already observed at the plot scale was also found to be significant at the provincial scale. The linkages between wheat yields and large scale climate have been analyzed for the first time over Morocco. In agreement with previous studies, results show a complex and competing influence of different climate phenomena. The NAO is found to be significantly related to yields during the early stage of wheat growth in December, whereas the SCA correlates with yields later in the season, in January and February. Interesting lagged correlations with higher lead time are also highlighted, with the leading modes of SST variability in the equatorial Atlantic during October (the “Atlantic Niño” mode) and in the North Atlantic (the “Atlantic tripole” mode) in February. Our conclusion is that regional climate indices and variables represent valuable information with which to increase lead time and skill regarding wheat yield predictions in Morocco.     

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.     

Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human

Background

Studies in mice have shown that PPARα is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARα in human liver. Here we set out to compare the function of PPARα in mouse and human hepatocytes via analysis of target gene regulation.

Methodology/Principal Findings

Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARα agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARα expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362–672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARα in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARα targets, including CPT1A, HMGCS2, FABP1, ACSL1, and ADFP. Several genes were identified that were specifically induced by PPARα in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARα targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8.

Conclusions/Significance

Our results suggest that PPARα activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARα as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARα regulates a mostly divergent set of genes in mouse and human hepatocytes.     

Pigment–Protein Complexes and the Number of Reaction Centers of the Photosystems in Pea Chlorophyll Mutants

We studied fluorescent and absorption properties of the chloroplasts and pigment–protein complexes isolated by gel electrophoresis from the leaves of pea, the parent cultivar Torsdag and mutants chlorotica 2004 and 2014. Specific fluorescence peaks of chlorophyll forms in individual complexes have been determined from the absorption and fluorescence spectra of the chloroplast chlorophyll and their second derivatives at 23 and –196°C. The mutant chlorotica 2004 proved to have an increased intensity of a long-wave band of the light-harvesting complex I at both 23°C (745 nm) and –196°C (728 nm). At the same time, this mutant manifested a decreased accumulation of the chlorophyll forms making up the nearest-neighbor antenna of the PS I reaction center (at 690, 697, and 708 nm). No spectral differences have been revealed between chlorotica 2014 mutant and the parent cultivar. Gel electrophoresis revealed the synthesis of all chlorophyll–protein complexes in both mutants. At the same time, analysis of photochemical activity of PS I and PS II reaction centers and calculations of their number and the size of the light-harvesting antenna have shown that the number of reaction centers in the PS I of chlorotica 2004 mutant is reduced by a factor of 1.7 because its chlorophyll a–protein complex is disturbed by the mutation. The primary effect of chlorotica 2014 mutation remains unclear. The proportional changes in the content of photosystem complexes in this mutant suggest that they are secondary and result from a 50% decrease in chlorophyll content.     

Analysis of DNA–Protein Contacts in a Complex between Methyltransferase SsoII and a Promoter Region of the SsoII Restriction–Modification Genes

Heterocyclic bases and phosphate groups involved in the DNA–methyltransferase SsoII (M·SsoII) interaction were identified in the regulatory DNA region localized within the promoter region of the SsoII restriction–modification genes by footprinting with the use of formic acid, hydrazine, dimethyl sulfate, and N-ethyl-N-nitrosourea as modifying agents. It has been established that the enzyme interacts with three guanines, one adenine, two thymines, and three phosphate groups of each strand of the DNA duplex. These heterocyclic bases and phosphate groups are disposed symmetrically within the 15-mer inverted repeat of the regulatory DNA region. It has been demonstrated by footprinting with dimethyl sulfate that the C7 atoms of guanines interacting with the enzyme are exposed to the DNA major groove. Two theoretical models were built describing the contacts in a complex between M·SsoII and the regulatory DNA region.     

Analysis of the Fungal Flora in Environmental Dust Samples by PCR–SSCP Method

Conventional microbiological techniques yield only limited information on the composition of fungal communities in dust. The aim of this study was to establish and optimize PCR-single strand conformation polymorphism (PCR–SSCP) analysis for investigation of fungal diversity in rural dust samples. Three different DNA extraction protocols were tested on 38 fungal cultures. A total of six known universal fungal primer pairs were tested targeting the 18S rRNA gene, the 28S rRNA gene and the ITS region, respectively. Objective evaluation was performed with respect to the following parameters: efficiency to amplify all 38 strains; separation of seven species from different phylogenetic groups on the SSCP gel; additional bands in PCR–SSCP analysis; possibility to classify the amplified gene fragments to species level. Primer ITS1/ITS4 and PowerSoil? DNA isolation showed the best performance in most cases and were chosen for further analysis. The detection limit of the developed system was 200 CFU/g dust. Moreover, the reproducibility of the system could be demonstrated, leading to average profile similarities of 94.94 % [SD = 2.51] within gels, 93.03 % [SD = 4.69] between different days and 87.66 % [SD = 6.62] between different gels when testing shed and mattress dust samples. Sequencing allowed identification on species level, in detail: Alternaria alternata, Cladosporium sphaerospermum, Cladosporium cladosporioides as well as the yeasts Candida cabralensis and Candida catenulata. This demonstrates the adaptability of the method. In this study, a standardized system for fungal community analysis was developed that provides reproducible results applicable for epidemiological purposes.     

Analysis and modeling of time-variant amplitude–frequency couplings of and between oscillations of EEG bursts

Low-frequency (0.5-2.5 Hz) and individually defined high-frequency (7-11 or 8-12 Hz; 11-15 or 14-18 Hz) oscillatory components of the electroencephalogram (EEG) burst activity derived from thiopental-induced burst-suppression patterns (BSP) were investigated in seven sedated patients (17-26 years old) with severe head injury. The predominant high-frequency burst oscillations (>7 Hz) were detected for each patient by means of time-variant amplitude spectrum analysis. Thereafter, the instantaneous envelope (IE) and the instantaneous frequency (IF) were computed for these low- and high-frequency bands to quantify amplitude-frequency dependencies (envelope-envelope, envelope-frequency, and frequency-frequency correlations). Time-variant phase-locking, phase synchronization, and quadratic phase couplings are associated with the observed amplitude-frequency characteristics. Additionally, these time-variant analyses were carried out for modeled burst patterns. Coupled Duffing oscillators were adapted to each EEG burst and by means of these models data-based burst simulations were generated. Results are: (1) strong envelope-envelope correlations (IE courses) can be demonstrated; (2) it can be shown that a rise of the IE is associated with an increase of the IF (only for the frequency bands 0.5-2.5 and 7-11 or 8-12 Hz); (3) the rise characteristics of all individually averaged envelope-frequency courses (IE-IF) are strongly correlated; (4) for the 7-11 or 8-12 Hz oscillation these associations are weaker and the variation between the time courses of the patients is higher; (5) for both frequency ranges a quantitative amplitude-frequency dependency can be shown because higher IE peak maxima are accompanied by stronger IF changes; (6) the time range of significant phase-locking within the 7-11 or 8-12 Hz frequency bands and of the strongest quadratic phase couplings (between 0.5-2.5 and 7-11 or 8-12 Hz) is between 0 and 1,000 ms; (7) all phase coupling characteristics of the modeled bursts accord well with the corresponding characteristics of the measured EEG burst data. All amplitude-frequency dependencies and phase locking/coupling properties described here are known from and can be discussed using coupled Duffing oscillators which are characterized by autoresonance properties.     

Analysis of the interactions between Eudragit® L100 and porcine pancreatic trypsin by calorimetric techniques

Flexible-chain polymers with charge (polyelectrolytes) can interact with globular proteins with a net charge opposite to the charge of the polymers forming insoluble complexes polymer-protein. In this work, the interaction between the basic protein trypsin and the anionic polyelectrolyte Eudragit^® L100 was studied by using isothermal calorimetric titrations and differential scanning calorimetry. Turbidimetric assays allowed determining that protein-polymer complex was insoluble at pH below 5 and the trypsin and Eudragit^® L100 concentrations required forming the insoluble complex. DSC measurements showed that the T_m and denaturalization heat of trypsin increased in the polymer presence and the complex unfolded according to a two-state model. ΔH° and ΔS° binding parameters obtained by ITC were positives agree with hydrophobic interaction between trypsin and polymer. However, ionic strength of 1.0 M modified the insoluble complex formation. We propose a mechanism of interaction between Eudragit^® L100 and trypsin molecules that involves both hydrophobic and electrostatic interactions. Kinetic studies of complex formation showed that the interaction requires less than 1 min achieving the maximum quantity of complex. Finally, a high percentage of active trypsin was precipitated (approximately 76% of the total mass of protein). These findings could be useful in different protocols such as a protein isolation strategy, immobilization or purification of a target protein.     

Computational Assessment of Protein–protein Binding Affinity by Reversely Engineering the Energetics in Protein Complexes

The cellular functions of proteins are maintained by forming diverse complexes. The stability of these complexes is quantified by the measurement of binding affinity, and mutations that alter the binding affinity can cause various diseases such as cancer and diabetes. As a result, accurate estimation of the binding stability and the effects of mutations on changes of binding affinity is a crucial step to understanding the biological functions of proteins and their dysfunctional consequences. It has been hypothesized that the stability of a protein complex is dependent not only on the residues at its binding interface by pairwise interactions but also on all other remaining residues that do not appear at the binding interface. Here, we computationally reconstruct the binding affinity by decomposing it into the contributions of interfacial residues and other non-interfacial residues in a protein complex. We further assume that the contributions of both interfacial and non-interfacial residues to the binding affinity depend on their local structural environments such as solvent-accessible surfaces and secondary structural types. The weights of all corresponding parameters are optimized by Monte-Carlo simulations. After cross-validation against a large-scale dataset, we show that the model not only shows a strong correlation between the absolute values of the experimental and calculated binding affinities, but can also be an effective approach to predict the relative changes of binding affinity from mutations. Moreover, we have found that the optimized weights of many parameters can capture the first-principle chemical and physical features of molecular recognition, therefore reversely engineering the energetics of protein complexes. These results suggest that our method can serve as a useful addition to current computational approaches for predicting binding affinity and understanding the molecular mechanism of protein–protein interactions.     

Introduction of Alien Host–parasite Complexes in a Natural Environment and the Symbiota Concept

When a species is introduced in a new territory its parasites may also follow, thus forming a sort of biotic unit. We propose the term symbiota to indicate this host–parasite complex. This concept is at the basis of the present research in which 11 species of alien and native freshwater fish were collected by electrofishing in the Po river area (Northern Italy). The alien host fish Ictalurus melas and Silurus glanis were parasitized by only one monogenean species each. Lepomis gibbosus, of North American origin, hosted four monogeneans and Carassius carassius three. All these parasites should be considered as alien species, except those collected from C. carassius that should be considered in part natives and in part aliens.     

The role of temperament in the relationship between morningness–eveningness and mood

Apart from differences in circadian phase position, individuals with different morningness–eveningness levels vary in many more characteristics. Particularly consistent relationships have been observed between morningness–eveningness and mood. Eveningness has been associated with disadvantageous mood, e.g. depressiveness in healthy individuals, and mood disorders. A concept of social jetlag suggests that evening subjects function in less advantageous environments due to discrepancies between internal and social time (societies promote morning-oriented functioning), which results in their lowered mood. Individual temperament, as defined by the Regulative Theory of Temperament (RTT), refers to the capacity of the human organism to meet environmental requirements – the greater the capacity, the less negative impact of external conditions. Thus, the aim of this study is to determine which RTT traits are linked to both morningness–eveningness and mood dimensions and to test whether they account for the relationship between morningness–eveningness and mood. A sample of 386 university students (267 female) aged between 19 and 47 (M?=?21.15, SD?=?4.23) years completed the University of Wales Institute of Science and Technology (UWIST) Mood Adjective Check List, Morningness–Eveningness Questionnaire and Formal Characteristics of Behaviour – Temperament Inventory. Analyses revealed lower endurance (EN) and higher emotional reactivity (ER) related to eveningness as well as to lower hedonic tone (HT), energetic arousal (EA) and to higher tense arousal (TA). Moreover, eveningness was associated with lower HT, EA and higher TA. Among RTT traits, EN was most strongly related to eveningness, and mediation analyses revealed that this temperamental trait fully mediated the relationship between eveningness and the three mood dimensions. The remaining RTT traits did not provide more explanation of the association between morningness–eveningness and mood than EN itself. If subjects did not differ in EN, the association between morningness–eveningness and mood was absent. EN is discussed as a protective factor against negative consequences of social jetlag and particularly lowered mood in evening individuals.     

Genetic Diversity of the Carrion and Jungle Crows as Evidenced by RAPD–PCR Analysis

RAPD–PCR analysis of the genetic diversity of the carrion crow (Corvus corone) and jungle crow (C. macrorhynchos) living in the continental parts of their species ranges and on some Russian and Japanese Far Eastern islands has been performed. Taxon-specific molecular markers have been found for each species. The genetic diversity of the carrion crow is considerably less than that of the jungle crow at the same genetic distance (P ₉₅ = 68.2%, D _N = 0.27 and P ₉₅ = 88.4%, D _N = 0.24, respectively). In both species, the genetic polymorphism of island samples is almost two times greater than that of continental samples (62 and 31.8%, respectively, for C. corone and 81.5 and 47.2%, respectively, for C. macrorhynchos). In addition, differences in genetic diversity between males and females (P ₉₅ = 55.1 and 72.1, respectively) has been found in the carrion crow but not in the jungle crow. The gene diversity of C. macrorhynchos is greater than that of C. corone: the mean numbers of alleles per locus are 2 and 1.81, effective numbers of alleles are 1.62 and 1.43, and the mean expected heterozygosities are 0.39 and 0.30, respectively. The phenograms and phylograms significantly segregate the clusters of the carrion and jungle crows. The clustering patterns of carrion crows corresponds to the intraspecies taxonomic and geographic differentiation: subspecies C. c. corone and C. c. orientalis living in the western and eastern parts of the species range, respectively, form different subclusters. The cluster of the jungle crow does not exhibit differentiation into subspecies C. m. mandshuricus and C. m. japonensis; molecular genetic differences between them are small.     

Marker Analysis of Quantitative Traits in Maize by ISSR–PCR

The segregating maize population (GK26 × Mo17)F₂ has been used for identification of ISSR markers able to reveal a significant difference between alleles by a quantitative index. Confidence ranges have been determined for variation in 17 quantitative traits. Variations in the traits under study correlate with the inheritance of 16 marker loci have been found. The nature of these correlations and the possibility of chromosomal mapping of genetic markers are discussed.     

Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC–ELSD to reveal accumulation differences of sugar and lipid

Biotechnology Letters - To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes...