Dietary CP and amino acid restriction has a different impact on the dynamics of protein,amino acid and fat deposition in entire male,castrated and female pigs

Breeding efforts over the last decades altered markedly empty body (EB) composition of pigs. This study aimed to re-evaluate the dynamics of changes in the composition and deposition rate of fat, protein and amino acids (AA) in the EB from birth to 140 kg BW depending on the dietary CP and AA supply in a current pig genotype. In the experiment 66 entire male, 58 castrated and 66 female Swiss Large White pigs were used. From 20 kg BW onwards, they had either ad libitum access to a control (C) diet or a diet (LP) compared to diet C only 80% of CP, lysine, methione+cystine, threonine and tryptophan. The EB composition was determined at birth on eight boars and eight females, at 10 and 20 kg BW on two boars, two castrates and two females, and at 20 kg intervals from 40 to 140 kg BW, on four pigs per gender and dietary treatment. Each EB fraction was weighed and analysed for protein, fat and AA profile. The AA-to-lysine ratio was calculated and the different chemical component contents were fitted to allometric regressions. Overall, C-boars had the greatest EB protein and AA content and deposition rates, and lowest fat content and deposition rates. At the beginning of the grower period, LP-castrates and females displayed the lowest protein and AA and the highest fat deposition rates. However, compared with their counterparts in the C-group, in LP-castrates and females protein and AA deposition rates were greater above 64 and 40 kg EB weight, respectively, whereas fat deposition rates was lower above 80 kg EB weight. Thus, there seems a great potential to optimise protein and AA efficiency especially in the finisher period in castrates and females. Important individual variations were found in the essential AA-to-lysine ratio of the EB. Phenylalanine and threonine-to-lysine ratios decreased with increasing EB weight. Valine- and threonine-to-lysine ratios in C-castrates and C-females were 5% and 4% greater than recently reported by the National Research Council (NRC) whereas cysteine-, methionine- and tyrosine-to-lysine ratios were lower by 34%, 25% and 10%, respectively. The clear differences found between the EB AA-to-lysine ratios in the present study and the NRC might partly be explained by the genotype and the temporal changes in the relative weight of each EB fraction or changes in the AA profile. Nevertheless, these findings on changes in the essential AA profile of tissue protein warrant further studies.     

Comparative expression analysis of heavy metal ATPase subfamily genes between Cd-tolerant and Cd-sensitive turnip landraces

The heavy metal ATPase(HMA)subfamily is mainly involved in heavy metal(HM)tolerance and transport in plants,but an understanding of the definite roles and mechanisms of most HMA members are still limited.In the present study,we identified 14 candidate HMA genes named BrrHMAl—BrrHMA8 from the turnip genome and analyzed the phylogeny,gene structure,chromosome distribution,and conserved domains and motifs of HMAs in turnip(Brassica rapa var.rapa).According to our phylogenetic tree,the BrrHMAs are divided into a Zn/Cd/Co/Pb subclass and Cu/Ag subclass.The BrrHMA members show similar structural characteristics within subclasses.To explore the roles of BrrHMAs in turnip,we compared the gene sequences and expression patterns of the BrrHMA genes between a Cd-tolerant landrace and a Cd-sensitive landrace.Most BrrHMA genes showed similar spatial expression patterns in both Cd-tolerant and Cd-sensitive turnip landraces;some BrrHMA genes,however,were differentially expressed in specific tissue in Cd-tolerant and Cd-sensitive turnip.Specifically,BrrHMA genes in the Zn/Cd/Co/Pb subclass shared the same coding sequence but were differentially expressed in Cd-tolerant and Cd-sensitive turnip landraces under Cd stress.Our findings suggest that the stable expression and up-regulated expression of BrrHMA Zn/Cd/Co/Pb subclass genes under Cd stress may contribute to the higher Cd tolerance of turnip landraces.     

An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.     

Identification of functional candidate genes for drought tolerance in rice

Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analyses using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Bin-Ying Fu and Jian-Hua Xiong are contributed to this work equally.     

Structural and functional properties of αβ-heterodimers of tropomyosin with myopathic mutations Q147P and K49del in the β-chain

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein that plays a key role in the Ca²⁺-regulated contraction of striated muscles. Two Tpm isoforms, α (Tpm 1.1) and β (Tpm 2.2), are expressed in fast skeletal muscles. These Tpm isoforms can form either αα and ββ homodimers, or αβ heterodimers. However, only αα-Tpm and αβ-Tpm dimers are usually present in most of fast skeletal muscles, because ββ-homodimers are relatively unstable and cannot exist under physiologic conditions. Nevertheless, the most of previous studies of myopathy-causing mutations in the Tpm β-chains were performed on the ββ-homodimers. In the present work, we applied different methods to investigate the effects of two myopathic mutations in the β-chain, Q147P and K49del (i.e. deletion of Lys49), on structural and functional properties of Tpm αβ-heterodimers and to compare them with the properties of ββ-homodimers carrying these mutations in both β-chains. The results show that the properties of αβ-Tpm heterodimers with these mutations in the β-chain differ significantly from the properties of ββ-homodimers with the same substitutions in both β-chains. This indicates that the αβ-heterodimer is a more appropriate model for studying the effects of myopathic mutations in the β-chain of Tpm than the ββ-homodimer which virtually does not exist in human skeletal muscles.     

Identification and characterization of carbapenem binding sites within the RND-transporter AcrB

Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-OprM, the major efflux pump transporter in Pseudomonas aeruginosa. However, not much is known regarding recognition and transport of these compounds by AcrAB-TolC, which is the MexAB-OprM homologue in Escherichia coli and by definition the paradigm model for structural studies on efflux pumps. Prompted by this motivation, we unveiled the molecular details of the interaction of imipenem and meropenem with the transporter AcrB by combining computer simulations with biophysical experiments. Regarding the interaction with the two main substrate binding regions of AcrB, the so-called access and deep binding pockets, molecular dynamics simulations revealed imipenem to be more mobile than meropenem in the former, while comparable mobilities were observed in the latter. This result is in line with isothermal titration calorimetry, differential scanning experiments, and binding free energy calculations, indicating a higher affinity for meropenem than imipenem at the deep binding pocket, while both sharing similar affinities at the access pocket. Our findings rationalize how different physico-chemical properties of compounds reflect on their interactions with AcrB. As such, they constitute precious information to be exploited for the rational design of antibiotics able to evade efflux pumps.     

Structural characterization of cationic DODAB bilayers containing C24:1 β-glucosylceramide

The effect of 5 mol%, 9 mol%, and 16 mol% of C24:1 β-glucosylceramide (βGlcCer) on the structure of cationic DODAB bilayers was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy and fluorescence microscopy. βGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed in fluorescence microscopy or ESR spectra. The latter showed that βGlcCer destabilized the gel phase of DODAB bilayers by decreasing the gel phase packing. As a consequence, βGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB bilayers, as seen in DSC thermograms. ESR spectra also showed that βGlcCer induced an increase in DODAB fluid phase order and/or rigidity. Despite their different structures, a similar effect of loosening the gel phase packing and turning the fluid phase more rigid/organized has also been observed when low molar fractions of cholesterol were incorporated in DODAB bilayers. The structural characterization of mixed membranes made of cationic lipids and glucosylceramides may be important for developing novel immunotherapeutic tools such as vaccine adjuvants.     

Gene co-expression network analysis for identifying genetic markers in Parkinson's disease - a three-way comparative approach

Parkinson's disease (PD) is a neurodegenerative disorder involving progressive deterioration of dopaminergic neurons. Although few genetic markers for familial PD are known, the etiology of sporadic PD remains poorly understood. Microarray data was analysed for induced pluripotent stem cells (iPSCs) derived from PD patients and mature neuronal cells (mDA) differentiated from these iPSCs. Combining expression and semantic similarity, a highly-correlated PD interactome was constructed that included interactions of established Parkinson's disease marker genes. A novel three-way comparative approach was employed, delineating topologically and functionally important genes. These genes showed involvement in pathways like Parkin-ubiquitin proteosomal system (UPS), immune associated biological processes and apoptosis. Of interest are three genes, eEF1A1, CASK, and PSMD6 that are linked to PARK2 activity in the cell and thereby form attractive candidate genes for understanding PD. Network biology approach delineated in this study can be applied to other neurodegenerative disorders for identification of important genetic regulators.     

Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the P(i) generated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) the V:(max) and K:(m) values of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of Mathematica 3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the influence of nM-microM concentrations of ATP on the metabolism of AMP.