Vitamin A metabolism in rats chronically treated with 3,3′,4,4′,5,′-hexabromobiphenyl

Chronic dietary administration of 3,3′,4,4′,5,5′-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA: retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-³H]retinyl acetate (10 μg), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.     

A validated HPLC method for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) with fluorescence detection in raw material and pill form: application to an in vitro dissolution test and a content uniformity test

A simple, sensitive and rapid HPLC method with fluorescence detection for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) in the raw material and pill form was developed. Liquid chromatography was performed on a C₁₈ column (250 × 4.6 mm i.d., 5 µm particle size), the mobile phase consisted of methanol and 0.05 M sodium dihydrogen phosphate buffer (80 : 20, v/v), and the apparent pH of the mobile phase was adjusted to 3. The fluorescence detector was operated at excitation/emission wavelengths of 275/400 nm. The proposed method allows the determination of DDB within concentration range 0.1–1.5 µg/mL with a limit of detection of 0.032 µg/mL, a limit of quantification of 0.097 µg/mL and a correlation coefficient of 0.9997. The proposed method has been successfully applied for the analysis of DDB in its pills with a percentage recovery of 98.45 ± 0.32. The method was fully validated according to ICH guidelines. Moreover, the high sensitivity of the method permits its use in an in vitro dissolution test for DDB under simulated intestinal conditions. In addition, the proposed method was extended to a content uniformity test according to USP guidelines. Copyright © 2014 John Wiley & Sons, Ltd.     

Age-related increases of 8-hydroxy-2′-deoxyguanosine and DNA–protein crosslinks in mouse organs

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and DNA–protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by ³²P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during ³²P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8±0.7 8-OH-dG/10⁵ normal nucleotides), brain (1.6±0.5) and heart (2.3±0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7±0.4; brain, 3.6±1.1; heart, 6.8±2.2 8-OH-dG/10⁵ normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P<0.01), and 3.0-fold increase in heart (P<0.001). A similar trend was observed for DPXL levels, which were the 1.8±0.3%, 1.2±0.2%, and 2.2±0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9±0.4%, 2.0±0.4%, and 3.4±0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P<0.01), and 1.5 (P<0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r=0.619, P<0.001) and heart (r=0.800, P<0.0001), but not in liver (r=0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.     

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates inhibit the repair of DNA in rat liver chromatin

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates are shown to be strong inhibitors of repair DNA synthesis in γ-irradiated rat liver chromatin. The activity of these compounds is comparable with that of the most effective inhibitor of the DNA polymerase β-catalyzed repair DNA synthesis.     

Molecular and crystal structure of an intercalation complex: Proflavine–cytidylyl-(3′,5′)-guanosine

The high-resolution crystal and molecular structure of a 3:2 complex of proflavine and cytidylyl-(3′,5′)-guanosine is described. The complex exhibits more than one mode of dye binding to the dinucleoside phosphate. One proflavine cation is symmetrically intercalated between the base pairs. The other proflavine cations and ones related by symmetry stack above and below the base pairs and also hydrogen bond externally to the duplex. The conformation of the CpG is most similar to A-RNA with all C(3′)-endo sugar puckering. To allow the base pairs to stretch from the normal 3.4-Å separation to a 6.8-Å separation, the torsion angles ? and χ of the guanosine are increased by about 60° from the values found in RNA. The crystal structure itself contains disordered sulfate anions and is highly solvated, with all but one water molecule involved in a continuous water–sulfate channel.     

Polarized fluorescence in an electric field: comparison with other electrooptical effects for rodlike fragments of DNA and the problem of the saturation of the induced moment in polyelectrolytes

Electrical birefringence, electrical dichroism and polarisation of fluorescence in an electric field experiments have been performed at high fields on sonicated fragments of DNA labelled with Acridine Orange. The latter electrooptical effect gives access to the field dependence of the fourth moment of the orientation function while the two former give access to the field dependence of the second moment. The origin of the large departure from an E2 dependence at rather low degrees of orientation is extensively discussed. Following a suggestion of Shirai on the calculation of orientational averages for a saturated induced moment, we can show that this model rationalizes the existence of a linear E dependence of the orientation factor at intermediate fields and explains very well our experimental results. When applied to previous dichroic data at higher fields it shows that the low value of the dichroism at saturation introduced to fit with other models, in contradiction with the absence of base tilting in the B form of DNA, is not required for a quantitative fit with this new orientation mechanism. The transition from an E2 dependence at low fields to an E dependence at intermediate fields gives an estimate of the field required for the saturation of the ionic polarisation E approximately 6 kV/cm.     

Theoretical probes of conformational fluctuations in S-peptide and RNase A/3′–UMP enzyme product complex

The dynamic properties of the RNase A/3′–UMP enzyme/product complex and the S-peptide of RNase A have been investigated by molecular dynamics simulations using suitable generalization of ideas introduced to probe the energy landscape in structural glasses. We introduce two measures, namely, the kinetic energy fluctuation metric and the force metric, both of which are used to calculate the time needed for sampling the conformation space of the molecules. The calculation of the fluctuation metric requires a single trajectory whereas the force metric is computed using two independent trajectories. The vacuum MD simulations show that for both systems the time required for kinetic energy equipartitioning is surprisingly long even at high temperatures. We show that the force metric is a powerful means of probing the nature and relative importance of conformational substates which determine the dynamics at low temperatures. In particular the time dependence of the non-bonded force metric is used to demonstrate that at low temperatures the system is predominantly localized hi a single cluster of conformational substates. The force metric is used to show that relaxation of long range (in sequence space) interactions must be mediated by a sequence of local dihedral angle transitions. We also argue that the time needed for compact structure formation is intimately related to the time needed for the relaxation of the dihedral angle degrees of freedom. The tame for non-bonded interactions, which drive protein molecules to fold under appropriate conditions, to relax becomes extremely long as the temperature is lowered suggesting that the formation of maximally compact structure hi proteins must be a very slow process. © 1993 Wiley-Liss, Inc.     

Polarized fluorescence in an electric field: steady state and transient values for the fourth moment of the orientation function at arbitrary fields

Steady state and time dependent expressions for the field dependence of the fourth moment of the orientation function at arbitrary fields, which are requested for the interpretation of polarisation of fluorescence in an electric field, have been obtained for permanent and induced dipole orientation. Some general features of the results and some problems connected with their application are pointed out.     

Mycobacterium tuberculosis class II apurinic/apyrimidinic‐endonuclease/3′‐5′ exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β‐clamp

The class‐II AP‐endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β‐clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel ₂₃₉QLRFPKK₂₄₅ motif in the DNA‐binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding‐groove (PBG) and the C‐terminal of β‐clamp located on different domains interact with XthA. The β‐clamp‐XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β‐clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β‐clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β‐clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β‐clamp is important for interactions with XthA, while the C‐terminal domain predominantly mediates functional interactions in the substrate's presence.     

Improvements in the application of the 4,4-N,N-Dimethylaminoazobenzene-4′-isothiocyanate micromethod to the sequence analysis of proteins

Different experimental conditions have been tested to improve the sequence determination of peptides and proteins by the DABITC (4,4-N,N-dimethylaminoazobenzene-4′-isothiocyanate) method and to facilitate automation in the analysis of the released 4,4-N,N-di-methylaminoazobenzene-4′-thiohydantoin derivatives (DABTHs). Conditions for a complete and rapid separation of all amino acid derivatives have been optimized by using different reversed-phase columns. The stability of the DABTHs in several water-organic solvent mixtures was determined by quantitative analysis and permitted the selection of the appropriate solvents for use in autosamplers. Also the amino acid side-products specific to individual residues which may be observed during thin-layer chromatography of DABTHs can be completely resolved by HPLC and are helpful for a safe assignment of the amino acid residues. The analytical procedures developed have been used to examine the influence of oxygen and detergents on the efficiency of the application of the DABITC manual micromethod on proteins. In the presence of oxygen the recovery of DABTHs is lower in most cases than when the operation is carried out in an inert atmosphere. The presence of a limited amount of detergents does not interfere in the HPLC analysis of DABTHs and, moreover, can increase the efficiency of the sequence analysis of proteins depending on their nature and concentration. In particular, it has been observed that sodium dodecyl sulfate at a concentration of 0.1 % can in some cases produce a threefold increase in the recovery of DABTHs.     

Enhanced polymerization of polar macromolecules by an applied electric field with application to mitosis

Numerous cellular processes are characterized by the rapid polymerization of protein molecules to form rod-like structures. Examples include the formation of spindle fibers from tubulin during cell division and the polymerization of actin into the actin filaments of the pseudopod in chemotaxis. It has been proposed that these proteins possess an electric dipole moment and that the onset of an internal electric field triggers polymerization. In this theoretical study, the relative probability of polymerization of a polar protein species is calculated in the presence and absence of an electric field. There is a significant enhancement of polymerization in the presence of an electric field, which increases as the size of the attachment site decreases. We conclude that a cytoplasmic pool of suitable proteins will rapidly polymerize if an electric field is applied, while remaining in a random configuration in the absence of a field. This mechanism is applied to the mitotic spindle structure, and by assuming that the spindle poles become oppositely charged during mitosis, a finite difference method is used to calculate the spindle structure at metaphase. Good agreement is obtained with experiment data.     

A mixture model approach to the mapping of quantitative trait loci in complex populations with an application to multiple cattle families.

A mixture model approach is presented for the mapping of one or more quantitative trait loci (QTLs) in complex populations. In order to exploit the full power of complete linkage maps the simultaneous likelihood of phenotype and a multilocus (all markers and putative QTLs) genotype is computed. Maximum likelihood estimation in our mixture models is implemented via an Expectation-Maximization algorithm: exact, stochastic or Monte Carlo EM by using a simple and flexible Gibbs sampler. Parameters include allele frequencies of markers and QTLs, discrete or normal effects of biallelic or multiallelic QTLs, and homogeneous or heterogeneous residual variances. As an illustration a dairy cattle data set consisting of twenty half-sib families has been reanalyzed. We discuss the potential which our and other approaches have for realistic multiple-QTL analyses in complex populations.     

Studies of DNA dumbbells. IV. Preparation and melting of a DNA dumbbell with the 16 base-pair sequence 5′G-T-A-T-C-C-C-T-C-T-G-G-A-T-A-C3′ linked on the ends by dodecyl chains

The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C¹²H²⁴ (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T₄ loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na⁺ and revealed, regardless of [Na ⁺], the duplex linked by dodecyl loops is more stable by at least 6°C than the same duplex linked by T₄ loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T₄ end loops are essentially two state in 25 and 55 mM Na⁺. In contrast, significant deviations from two-state behavior were observed in 85 m MNa⁺. From our previously published melting data of DNA dumbbells with T_n end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881–892] and the dumbbell with T₄ end loops of this study, a plot of d(T_m)/d ln [Na⁺] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (T_m)/d ln [Na⁺] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue. © 1993 John Wiley & Sons, Inc.     

Mitotic DNA damage targets the Aurora A/TPX2 complex

We have previously shown that the DNA damage-induced G2 arrest is contributed by inhibition of Aurora A (AurA) and that transduction of active AurA into arrested cells allows bypassing the block through reactivation of CDK1. In this study, we investigated the mechanism of DNA damage-induced AurA inhibition. We provide evidence that ionizing radiation (IR) administered in mitosis, a time when AurA protein and enzymatic activity reach peak levels, impairs interaction with the partner TPX2, leading to inactivation of the kinase through dephosphorylation of AurA T-loop residue, T₂₈₈. We find that decreased AurA-TPX2 complex formation in response to irradiation results from reduced cellular levels of TPX2, an effect that is both contributed by increased APC/CDH1-dependent protein degradation and decreased translation of TPX2 mRNA.     

Structure and properties of a novel OH bridged dimetal helical complex with 6,6-dimethyl-2,2′:6′,2″:6″,2:6,2-quinquepyridine ligand

A new monohelical OH⁻ bridged dinuclear complex [Zn₂(dmqpy)(OOCCH₃)₂(μ-OH)][ClO₄] · 0.5EtOH, where dmqpy is 6,6-dimethyl-2,2′:6′,2″:6″,2:6,2-quinquepyridine, has been synthesized and characterized by X-ray crystallography: monoclinic, space group P2₁/c, a=13.670(1), b=14.751(1), c=16.782(1) Å, β=96.59(1)°, U=3361.7(4) ų, Z=4, R=0.0601. Two Zn(II) ions are in different coordination modes, one is five-coordinate with a N₃O₂ donor set and the other is N₂O₂ four-coordinate with a distorted tetrahedral geometry, and the zinc ions are bridged by a hydroxyl group. The presence of the OH⁻ bridge is further confirmed by electrospray mass and infrared spectroscopies. The solution properties of the complex were investigated by ¹H NMR spectroscopy. The results of NMR indicate that the complex has higher symmetry in solution than in the solid state.     

Interactions of vasopressin and oxytocin receptors with vasopressin analogues substituted in position 2 with 3,3′‐diphenylalanine – a molecular docking study

Vasopressin and oxytocin receptors belong to the superfamily of G protein‐coupled receptors and play an important role in many physiological functions. They are also involved in a number of pathological conditions being important drug targets. In this work, four vasopressin analogues substituted at position 2 with 3,3′‐diphenylalanine have been docked into partially flexible vasopressin and oxytocin receptors. The bulky residue at position 2 acts as a structural restraint much stronger in the oxytocin receptor (OTR) than in the vasopressin V2 receptor (V2R), resulting in a different location of the analogues in these receptors. This explains the different, either agonistic or antagonistic, activities of the analogues in V2R and OTR, respectively. In all complexes, the conserved polar residues serve as anchor points for the ligand both in OTR and V2R. Strong interactions of the C‐terminus of analogue II ([Mpa¹,d ‐Dpa²,Val⁴,d ‐Arg⁸]VP) with extracellular loop 3 may be responsible for its highest activity at V2R. It also appears that V2R adapts more readily to the docking analogues by conformational changes in the aromatic side chains triggering receptor activation. A weak activity at V1a vasopressin receptor appears to be caused by weak receptor–ligand interactions. Results of this study may facilitate a rational design of new analogues with the highest activity/selectivity at vasopressin and OTRs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Variation in the intensity of scattered light by solutions of DNA subjected to an electric field

Preliminary experiments have been carried out which measure the variation in the intensity of the light scattered by DNA solutions under the influence of an electric field. Data have been collected on the length of DNA molecules, their electric polarizability, and their dispersion behavior.