Coordinated regulation and colocalization of αv integrin and its activating enzyme proprotein convertase PC5 in vivo

Integrin alphav is involved in intracellular-extracellular signaling important for cytoskeleton alterations and control of cell movement. In vitro experiments indicate that the integrin alphav-subunit undergoes post-translational endoproteolytic cleavage. This type of activation requires the presence of suitable kexin/subtilisin-like proprotein convertases. In vitro experiments have demonstrated that, among several proprotein convertases, PC5A, and to a threefold lesser extent furin, can activate alphav integrin. The biological significance of these in vitro data would be further supported by a coexpression and coordinated regulation of the gene expression of alphav integrin and its activating enzyme PC5 in vivo. In the present study we investigated the regulation of alphav integrin and PC5 following balloon injury in vivo. Comparative immunocytochemistry revealed a coordinated regulation of alphav integrin and PC5 during vascular remodeling in rodents. Integrin alphav was found to be upregulated in PCNA-positive, proliferating vascular smooth muscle cells. Northern blots revealed no significant regulation of furin mRNA, whereas PC5A mRNA increased during vascular remodeling, suggesting that PC5 is the major convertase during neointima formation in vivo. Incubation of vascular smooth muscle cells with the Golgi-disturbing agent brefeldin A inhibited alphav integrin maturation, indicating that endoproteolytic cleavage occurs in the trans-Golgi network, were PC5 is localized. Thus, the present study further supports the concept that activation of alphav integrin occurs in the trans-Golgi network in vascular smooth muscle cells and involves PC5.     

The nature of conformational correlations in alpha- and beta-anomers of nucleic acid components in aqueous solution by nuclear magnetic resonance

The solution distribution of combinations of the sugar ring puckering domains, C2'endo(S), C3'endo(N), and C4'-C5' rotamers, +sc(g+), ap(t), -sc(g-), in alpha and beta-anomers in ribo- and deoxyribo- pyrimidine nucleic acid components can be determined from vicinal coupling constants (M. Remin, J. Biomol. Str. Dyn. 2, 211 (1984). A general correlation pattern with a conformational constant lambda reflecting an intrinsic physical property of the sugar-side chain ensemble, is developed and expressed in terms of four principles: I) The +sc rotamer contributes to the C3'endo population to a higher extent (1-Yt) than to C2'endo, (1-Yt-Yg-/Xs). II) The ap rotamer contributes to both C2'endo and C3'endo populations to the same extent (Yt). III) The -sc rotamer contributes only to the C2'endo population, (Yg-/Xs). IV) The molar fractions Xs, Yt and Yg- of conformations C2'endo, ap and -sc, respectively, are strongly correlated, lambda = (Yg-/Xs)/Yt approximately 0.5, and therefore Yt is a basic variable parameter which determines all others in the correlation pattern. In alpha-anomers, regardless of the type and conformation of the sugar ring and base, the molar fraction Yt = 0.37 +/- 0.02. This finding means that different alpha-anomers show one correlation pattern free of the influence of the base. In beta-anomers, structure and conformation of the base are important factors which modulate (through Yt) the correlation pattern, conserving its fundamental features. Yt is considerably increased by a syn-oriented pyrimidine base, but decreases when the base is anti. The transition from anti to syn orientation of the base is followed by destabilization of (C2'endo, +sc) in favor of (C3'endo, ap). The principles of conformational correlations rationalize a variety of correlations observed in the past.     

Effects of data incompleteness on the relative performance of parsimony and Bayesian approaches in a supermatrix phylogenetic reconstruction of Mustelidae and Procyonidae (Carnivora)

Missing data are commonly thought to impede a resolved or accurate reconstruction of phylogenetic relationships, and probabilistic analysis techniques are increasingly viewed as less vulnerable to the negative effects of data incompleteness than parsimony analyses. We test both assumptions empirically by conducting parsimony and Bayesian analyses on an approximately 1.5 × 10⁶‐cell (27 965 characters × 52 species) mustelid–procyonid molecular supermatrix with 62.7% missing entries. Contrary to the first assumption, phylogenetic relationships inferred from our analyses are fully (Bayesian) or almost fully (parsimony) resolved topologically with mostly strong support and also largely in accord with prior molecular estimations of mustelid and procyonid phylogeny derived with parsimony, Bayesian, and other probabilistic analysis techniques from smaller but complete or nearly complete data sets. Contrary to the second assumption, we found no compelling evidence in support of a relationship between the inferior performance of parsimony and taxon incompleteness (i.e. the proportion of missing character data for a taxon), although we found evidence for a connection between the inferior performance of parsimony and character incompleteness (i.e. no overlap in character data between some taxa). The relatively good performance of our analyses may be related to the large number of sampled characters, so that most taxa (even highly incomplete ones) are represented by a sufficient number of characters allowing both approaches to resolve their relationships. © The Willi Hennig Society 2009.     

Liposomal encapsulation enhances and prolongs the anti-inflammatory effects of water-soluble dexamethasone phosphate in experimental adjuvant arthritis

Introduction

The objective of this study was to evaluate the efficacy of intravenous (i.v.) injection of liposomally encapsulated dexamethasone phosphate (DxM-P) in comparison to free DxM-P in rats with established adjuvant arthritis (AA). This study focused on polyethylene glycol (PEG)-free liposomes, to minimize known allergic reactions caused by neutral PEG-modified (PEG-ylated) liposomes.

Methods

Efficacy was assessed clinically and histologically using standard scores. Non-specific and specific immune parameters were monitored. Activation of peritoneal macrophages was analyzed via cytokine profiling. Pharmacokinetics/biodistribution of DxM in plasma, synovial membrane, spleen and liver were assessed via mass spectrometry.

Results

Liposomal DxM-P (3 × 1 mg/kg body weight; administered intravenously (i.v.) on Days 14, 15 and 16 of AA) suppressed established AA, including histological signs, erythrocyte sedimentation rate, white blood cell count, circulating anti-mycobacterial IgG, and production of interleukin-1beta (IL-1β) and IL-6 by peritoneal macrophages. The suppression was strong and long-lasting. The clinical effects of liposomal DxM-P were dose-dependent for dosages between 0.01 and 1.0 mg/kg. Single administration of 1 mg/kg liposomal DxM-P and 3 × 1 mg/kg of free DxM-P showed comparable effects consisting of a partial and transient suppression. Moreover, the effects of medium-dose liposomal DxM-P (3 × 0.1 mg/kg) were equal (in the short term) or superior (in the long term) to those of high-dose free DxM-P (3 × 1 mg/kg), suggesting a potential dose reduction by a factor between 3 and 10 by liposomal encapsulation. For at least 48 hours after the last injection, the liposomal drug achieved significantly higher levels in plasma, synovial membrane, spleen and liver than the free drug.

Conclusions

This new PEG-free formulation of macrophage-targeting liposomal DxM-P considerably reduces the dose and/or frequency required to treat AA, with a potential to enhance or prolong therapeutic efficacy and limit side-effects also in the therapy of rheumatoid arthritis. Depot and/or recirculation effects in plasma, inflamed joint, liver, and spleen may contribute to this superiority of liposomally encapsulated DxM-P.     

Bovine oviductal fluid (bOF) collected in the follicular or luteal phase of the estrous cycle exerts similar effects on ram sperm kinematics and acrosome reactivity in vitro

This study examined the effects of bovine oviductal fluid (bOF) obtained during the follicular or luteal phase of the estrous cycle on ram sperm kinematics, capacitation status and plasma membrane (PM) integrity at various time points during the 24-h incubation period. Fresh ram spermatozoa were selected using the swim-up technique and then incubated separately with either follicular phase (FbOF) or luteal phase (LbOF) bovine oviductal fluid added to Fert-TALP medium (positive control - POSControl) or in Fert-TALP medium without capacitating agents (negative control - NEGControl) at 38 °C under 5% CO₂. Incubation with FbOF or LbOF for 2 h and 4 h promoted an increase (P < 0.05) in most of the sperm motility parameters as compared with the NEGControl group, and bOF-induced changes in sperm kinematics were similar (P > 0.05) to those seen in the POSControl group. After 6 h of incubation, the stimulatory effect of FbOF or LbOF on ram sperm kinematics was no longer observed (P > 0.05). Sperm PM integrity was not affected (P > 0.05) by incubation in bOF-supplemented media or in absence of capacitating factors (NEGControl). Although neither FbOF nor LbOF had any effect on sperm capacitation rates, the proportion of acrosome-reacted spermatozoa was greater (P < 0.05) for bOF-containing media compared with the NEGControl group during the long incubation periods (18 h and 24 h). In conclusion, bOF from either follicular or luteal phase of the estrous cycle enhances ram sperm motility for up to 4 h and the rate of acrosome reaction after long (18–24 h) incubation periods without affecting sperm viability.     

Increased Nigral Iron Content and Alterations in Other Metal Ions Occurring in Brain in Parkinson's Disease

Levels of iron, copper, zinc, manganese, and lead were measured by inductively coupled plasma spectroscopy in parkinsonian and age-matched control brain tissue. There was 31-35% increase in the total iron content of the parkinsonian substantia nigra when compared to control tissue. In contrast, in the globus pallidus total iron levels were decreased by 29% in Parkinson's disease. There was no change in the total iron levels in any other region of the parkinsonian brain. Total copper levels were reduced by 34-45% in the substantia nigra in Parkinson's disease; no difference was found in the other brain areas examined. Zinc levels were increased in substantia nigra in Parkinson's disease by 50-54%, and the zinc content of the caudate nucleus and lateral putamen was also raised by 18-35%. Levels of manganese and lead were unchanged in all areas of the parkinsonian brain studied when compared to control brains, except for a small decrease (20%) in manganese content of the medial putamen. Increased levels of total iron in the substantia nigra may cause the excessive formation of toxic oxygen radicals, leading to dopamine cell death.     

Determination of the Complete Correlation Between the Sugar Ring Puckers and 5′-Exocyclic Group Rotamers in Conformationally-Flexible Nucleic Acid Components from NMR Study

Abstract

Inspection of stereochemical models suggests a possible correlation between the proportion (Y_g-/Y_t) of the g^? and t rotamers and the S pucker populations irrespective of the anti-syn conformational composition of the base. Interpretation of the NMR vicinal coupling constants in terms of conformational populations shows a decline of Y_g-/Y_t with X_s approaching zero, consistent with high unfavorability of the Ng^? conformational combination in solution, a result supported by a X-ray crystallographic data survey. Hence, the underlying assumption introduced into the present study is that the g^? rotamer and the N pucker do not coexist together in solution. Therefore, the limiting value of Y_g-/Y_t corresponding to the S pucker could be determined for each compound individually. Finally, populations and relative free energies of all conformational combinations of Ng⁺, Nt, Sg⁺, St and Sg^? (except Ng^? which is not important) have been estimated.

Results of the present study suggest several interesting regularities concerning the syn-anti effect on populations and energies of the conformational combinations in ribo- and deoxyribo-nucleosides. (a) In the anti-type nucleosides, the Ng⁺ conformation is about 2 kJ/mol more stable than Nt, but in the syn-type, the Ng⁺ and Nt have comparable energy, (b) No important changes are observed in the Ng⁺ population comparing the anti-type and syn-type of ribo- and deoxyribo-nucleosides separately, (c) The Nt is considerable stabilized and simultaneously the Sg⁺ is strongly destabilized in the syn-type nucleosides relative to the anti-type, (d) Irrespective of the syn-anti composition the St is always more stable (1–2 kJ/mol) than the Sg^? conformational combination.     

1H NMR conformational study of antiherpetic C5-substituted 2'-deoxyuridines: insight into the nature of structure-activity relationships

1H NMR study and conformational analysis of a broad series of biologically important C5-substituted 2'-deoxyuridines, including alkyl, halogen, vinyl, hydroxymethyl, and hydroxy derivatives as well as nitro, formyl, trifluoromethyl, and dimethylamino substituents, is presented. A thorough analysis of chemical shifts in correlation with C5-substituent electronegativity as well as calculations by SCF semi-empirical method of the formal charge localized on C6 carbon is discussed in terms of charge distribution for electron attracting and electron donating groups. Conformation of the sugar ring is determined from proton-proton coupling constants and described in terms of pseudorotation between two main puckering domains C2'endo (S) and C3'endo (N). Generally, electron donating groups destabilise the N conformation, simultaneously decreasing the mean pseudorotation amplitude. Absolute assignments of the H5' and H5' methylene protons in 1H NMR spectra permitted the unequivocal determination of molar fractions of the three classical exocyclic C4'-C5' rotamers gauche+, trans, and gauche-, and correlation of them with the sugar ring puckering domains. Conformation about the glycosidic bond is described in terms of equilibrium between two conformational regions, anti and syn. Finally, the role of the C5-substituent in the creation of cytotoxic activity is considered on the basis of a simplified model assuming that compound activity is a function of substituent polar surface, its molecular volume, and its molecule polarity defined at the relative partition of the polar atoms.     

Systematics,evolution, and biogeography of the Pliocene stem meline badger Ferinestrix (Carnivora: Mustelidae)

Ferinestrix vorax is an extinct mustelid carnivoran of enigmatic relationships, known from a partial mandible and femur collected from the 3.2‐ and 3.6‐Myr‐old deposits of Hagerman Fossil Beds National Monument, Idaho, USA. Here, we report Ferinestrix rapax sp. nov. based on 80 remains of skull and dentition from a 3.1–3.6‐Myr‐old deposit of Udunga, Transbaikal, Russia. We demonstrate that Ferinestrix is a stem genus of the badger subfamily Melinae. This genus is distinctly larger and more carnivorous than any other total‐clade meline. We show that Ferinestrix originated in Asia and immigrated to North America no later than at the early (Zanclean) to late (Piacenzian) Pliocene transition, and that the North American F. vorax and Asian F. rapax underwent parallel evolution toward increased carnivory. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 167 , 208–226.