The synthesis and NMR investigation on novel boron derivatives of stavudine

Preparation and spectroscopic properties of novel boron-containing derivatives of anti-HIV agent stavudine are presented, The new compounds, (5′-O-(4,4,5,5-tetramethyl-1,3,2-dioxaboronate)-2′-3′-didehydro-2′-3′-dideoxythymidine and 5′-O-(dihydroxyboronate)-2′-3′-didehydro-2′-3′-dideoxythymidine), were prepared by direct reaction between stavudine and reagents containing BH moieties - pinacolborane and borane-dimethylsulfide complexes, respectively. The boron coordination equilibrium of those compounds was analyzed by water titration monitored by NMR. Results of the DFT calculations and NMR experiments pointed to structural and electronic similarity of tetrahedral boron complexes to phosphate group.     

Structure of Adenovirus Type 21 Knob in Complex with CD46 Reveals Key Differences in Receptor Contacts among Species B Adenoviruses

The complement regulation protein CD46 is the primary attachment receptor for most species B adenoviruses (Ads). However, significant variability exists in sequence and structure among species B Ads in the CD46-binding regions, correlating with differences in affinity. Here, we report a structure-function analysis of the interaction of the species B Ad21 knob with the two N-terminal repeats SCR1 and SCR2 of CD46, CD46-D2. We have determined the structures of the Ad21 knob in its unliganded form as well as in complex with CD46-D2, and we compare the interactions with those observed for the Ad11 knob-CD46-D2 complex. Surface plasmon resonance measurements demonstrate that the affinity of Ad21 knobs for CD46-D2 is 22-fold lower than that of the Ad11 knob. The superposition of the Ad21 and Ad11 knob structures in complex with CD46-D2 reveals a substantially different binding mode, providing an explanation for the weaker binding affinity of the Ad21 knob for its receptor. A critical difference in both complex structures is that a key interaction point, the DG loop, protrudes more in the Ad21 knob than in the Ad11 knob. Therefore, the protruding DG loop does not allow CD46-D2 to approach the core of the Ad21 knob as closely as in the Ad11 knob-CD46-D2 complex. In addition, the engagement of CD46-D2 induces a conformational change in the DG loop in the Ad21 knob but not in the Ad11 knob. Our results contribute to a more profound understanding of the CD46-binding mechanism of species B Ads and have relevance for the design of more efficient gene delivery vectors.The 52 human adenovirus (Ad) serotypes are divided into seven species (species A to G) (20). Species B Ads are of interest, as they cause severe infections of the respiratory tract, urinary tract, and kidney as well as multiorgan system failure and death in immunocompromised patients (2, 23, 24). Species B Ads can be further grouped into subspecies B1 (Ad3, Ad7, Ad16, Ad21, and Ad50) and subspecies B2 (Ad11, Ad14, Ad34, and Ad35). Viruses in the two subspecies differ in their tropisms: while most B1 viruses cause ocular and/or acute respiratory tract infections, the B2 viruses primarily cause persistent infections of the urinary tract as well as eye infections, meningitis, and infections of the gastrointestinal tract (10, 11, 43). The subspecies B1 Ad21, which is the subject of this study, recently caused outbreaks of acute respiratory disease (28).Adenoviruses have a nonenveloped icosahedral capsid with a linear double-stranded DNA (46). The major capsid proteins are the hexon, the penton base, and the fiber. The trimeric fiber protein, which protrudes from each of the 12 capsid vertices, consists of three distinct domains: an N-terminal tail, an elongated shaft, and a globular knob. The knob mediates cellular attachment to the primary receptors CD46 (16, 26, 38), coxsackievirus and adenovirus receptor (CAR) (36), and sialic acid (3). Virus attachment is followed by internalization into the host cell via clathrin-coated endocytosis and macropinocytosis, triggered by αv integrins (17, 27, 45).The species B adenovirus receptor CD46 is a member of a family of proteins that regulate complement activation and are constructed mainly from short consensus repeat (SCR) domains (25). The extracellular portion of CD46 contains four such domains (SCR1 to SCR4), and structural and functional analyses have established that interactions with Ad knobs require only the SCR1 and SCR2 domains (34). The four SCR domains are followed by a 25-amino-acid sequence that is rich in serine, threonine, and proline (the STP region); a single transmembrane segment; and a short cytoplasmic tail. Structural information is currently limited to the N-terminal CD46 domains SCR1 and SCR2. The crystal structure of a fragment comprising these two domains revealed a pronounced kink between the two repeats and some flexibility at the domain interface (7). The protein is expressed on all human cells with the exception of erythrocytes. CD46 acts as a cofactor for factor I, a serine protease that blocks further recruitment of the membrane attack complex by cleaving C3b and C4b (25, 40).CD46 is also a receptor for many other pathogens, including measles virus, human herpesvirus 6, Neisseria gonorrhoeae, Neisseria meningitidis, and group A streptococci (8, 13, 22, 30, 37).The structural analysis of the Ad11 knob in complex with the SCR1-SCR2 fragment of CD46 (CD46-D2) showed that engagement by the knob triggers a conformational change in CD46, producing an elongated, nearly linear conformation that differs substantially from the kinked conformation of the unliganded receptor (34). Furthermore, the structure of this complex provided an explanation for the previously observed critical role of Ad11 knob residue Arg279 in CD46 binding. Earlier mutagenesis studies demonstrated that a mutation of Arg279 to glutamine abolishes binding to CD46-D2 (18). Although the structure of the complex showed that Arg279 does not contact the receptor directly, its side chain lies parallel to that of the CD46-contacting residue Arg280. Stacking interactions between the guanidinium groups of Arg279 and Arg280, resulting in an arginine sandwich, likely play a central role in determining receptor specificity (33), and the mutation of Arg279 is thought to prevent Arg280 from forming contacts with CD46 (18).Although it also uses CD46 as a receptor, the Ad21 knob does not contain an arginine sandwich, as the residue corresponding to Arg279 in Ad11 is a serine. Therefore, the mode of binding of the Ad21 knob to CD46 is likely distinct from that observed for Ad11, consistent with the observation that previously reported mutational studies failed to determine a central binding motif among species B Ads (32, 44). The Ad21 and Ad11 knobs exhibit low sequence identity, especially at the surface loops that mediate binding to CD46 in Ad11. We therefore used a combination of structural and functional studies to establish the mechanism of Ad21 knob binding to CD46-D2. Our structural analysis reveals substantial differences in the numbers and types of contacts between the complexes of Ad11 and Ad21 with CD46-D2 and also in the relative orientations of CD46-D2 and its contacting knobs. Furthermore, our analysis allows the comparison of structural features of the Ad21 and Ad35 knobs, which are closely related in sequence yet also display significantly different CD46-D2-binding properties as well as different tissue tropisms. Thus, our findings result in a significantly enhanced understanding of the interactions between species B Ads and CD46.In addition to their role as pathogens, species B Ads serve as widely used gene delivery vectors, as they can transduce a broad range of possible target cells that are normally poorly permissive to other Ads, such as hematopoietic stem cells, dendritic cells, and malignant tumor cells (19, 41). Therefore, our results should also guide efforts to improve the gene delivery properties of these viruses.     

Association analysis of the E-selectin 98G > T polymorphism and the risk of childhood ischemic stroke

Genes related to platelet and arterial endothelial function have been recently considered as independent risk factors for stroke. We aimed to analyze a relationship between the E‐selectin 98G > T polymorphism and stroke in children and to observe the transmission of E‐selectin alleles from heterozygous parents to their affected children. We studied 59 children after stroke, 112 parents, and 87 healthy children. The E‐selectin 98G > T polymorphism was analyzed with the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. The frequency of the 98T allele in patients was almost twofold lower than in controls (5.1% vs. 9.8%, p = 0.145, odds ratios (OR) = 0.49) as well as carriers of the 98T allele (19.5% in controls vs. 8.5% in cases, p = 0.067, OR = 0.38). The G allele of the E‐selectin 98G > T polymorphism was more frequently transmitted to the children after stroke compared to the T allele (68% vs. 32%). In conclusion, we did not confirm the relationship between the 98G > T polymorphism of the E‐selectin gene and childhood ischemic stroke. There is still a need for further studies. Copyright © 2010 John Wiley & Sons, Ltd.     

The TRAF2 and TRAF6 expression in myomas and myometrium of women in reproduction and perimenopausal age

Uterine myomas represent one of the most common female diseases. Uterine myomas or fibromas are benign, hormone-responding tumours of, respectively, smooth muscles and fibroblasts and their aetiology induces a significant interest. In myomas the presence of aromatase was detected and, in addition, oestrogen was found to be synthesized in myoma cells. The studies were performed on myoma patients of generative age and those in peri-menopausal age. Expression of TRAF2 and TRAF6 proteins was examined using immunohistochemistry and Western blot approach in small and large uterine myomas isolated from women of various age. In addition, the evaluation was conducted at the periphery of every myoma. We indicated that the level of both tested proteins in myomas is higher than in control. TRAF2 level in myometrium was lower than in myomas but higher than in control. In the case of TRAF6 those changes were ambiguous. Age didn't have influence the level of expression in both tested TRAF in studied structures.     

Design,synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2′-β-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10 nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2′-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.     

Grass,mosses, algae,or leaves? Food preference among shredders from open-canopy streams

Shredder feeding is a vital process in making decomposition products available to biota in streams. To investigate which food sources shredders in open-canopy streams exploit, we conducted a feeding preference experiment with the invertebrate detritivores Limnephilus bipunctatus and Nemoura sp., which are commonly found in open-canopy streams on the Swedish island of Öland in the southern Baltic Sea. Leaves of birch, Swedish whitebeam, and shrubby cinquefoil; dead and fresh grass; water moss; and algae were offered to the shredders in multi- and single treatments. We hypothesized that food with high nutritional value would be preferred. Both taxa preferred leaves of shrubby cinquefoil, a bush common in the riparian zone of Öland streams; additionally Nemoura sp. also chose algae. Dead grass, the most abundant food source in the streams during the whole year, was the least consumed food type. The fresh food types had highest nutritional value, measured as carbon to nitrogen content. Therefore, food quality could not alone explain the preference of shrubby cinquefoil. However, among the detritus type offered, shrubby cinquefoil had the highest nutritional value. Shrubby cinquefoil may constitute one important energy source to these open-canopy stream ecosystems and may be essential in maintaining an abundant shredder community in these streams. Thus, the results of this study indicate that detrital resources are indeed important in open-canopy stream systems.     

Flow cytometric sorting and analysis of human epidermal stem cell candidates

ESC (epidermal stem cells) play a central role in the regeneration of human epidermis. These cells are also responsible for wound healing and neoplasm formation. Efficient isolation of ESC allows their use in medicine and pharmacy as well as in basic science. Cultured keratinocytes and ESC may be used as biological dressing in burn injuries, chronic wounds and hereditary disorders. Therefore, the isolation and characterization of ESC have been goals in biomedical science. Here, we present a flow cytometric method for the isolation and analysis of human ESC candidates. The strategy presented for the isolation of ESC combines previously proposed enzymatic digestion and FACS‐sorting of the obtained cell suspension that utilizes morphological features, integrin‐β1 expression and Rh123 (Rhodamine 123) accumulation of the cells. We also performed a flow cytometric analysis of sorted cells using a cell tracer.     

Clonal Characterization of Rat Muscle Satellite Cells: Proliferation,Metabolism and Differentiation Define an Intrinsic Heterogeneity

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨ_m), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.