The effect of the trabecular microstructure on the pullout strength of suture anchors

This study investigates how the microstructural properties of trabecular bone affect suture anchor performance. Seven fresh-frozen humeri were tested for pullout strength with a 5 mm Arthrex Corkscrew in the greater tuberosity, lesser tuberosity, and humeral head. Micro-computed tomography analysis was performed in the three regions of interest directly adjacent to individual pullout experiments. The morphometric properties of bone mineral density (BMD), structural model index (SMI), trabecular thickness (TbTh), trabecular spacing (TbS), trabecular number (TbN), and connectivity density were compared against suture anchor pullout strength. BMD (r=0.64), SMI (r=?0.81), and TbTh (r=0.71) showed linear correlations to the pullout strength of the suture anchor with p-values<0.0001. A predictive model was developed to explain the variances in the individual BMD, SMI, and TbTh correlations. The multi-variant model of pullout strength showed a stronger relationship (r=0.86) compared to the individual experimental results. This study helps confirm BMD is a major influence on the pullout strength of suture anchors, but also illustrates the importance of local microstructure in pullout resistance of suture anchors.     

Neuronal c-Jun is required for successful axonal regeneration, but the effects of phosphorylation of its N-terminus are moderate

Although neural c-Jun is essential for successful peripheral nerve regeneration, the cellular basis of this effect and the impact of c-Jun activation are incompletely understood. In the current study, we explored the effects of neuron-selective c-Jun deletion, substitution of serine 63 and 73 phosphoacceptor sites with non-phosphorylatable alanine, and deletion of Jun N-terminal kinases 1, 2 and 3 in mouse facial nerve regeneration. Removal of the floxed c-jun gene in facial motoneurons using cre recombinase under control of a neuron-specific synapsin promoter (junΔS) abolished basal and injury-induced neuronal c-Jun immunoreactivity, as well as most of the molecular responses following facial axotomy. Absence of neuronal Jun reduced the speed of axonal regeneration following crush, and prevented most cut axons from reconnecting to their target, significantly reducing functional recovery. Despite blocking cell death, this was associated with a large number of shrunken neurons. Finally, junΔS mutants also had diminished astrocyte and microglial activation and T-cell influx, suggesting that these non-neuronal responses depend on the release of Jun-dependent signals from neighboring injured motoneurons. The effects of substituting serine 63 and 73 phosphoacceptor sites (junAA), or of global deletion of individual kinases responsible for N-terminal c-Jun phosphorylation were mild. junAA mutants showed decrease in neuronal cell size, a moderate reduction in post-axotomy CD44 levels and slightly increased astrogliosis. Deletion of Jun N-terminal kinase (JNK)1 or JNK3 showed delayed functional recovery; deletion of JNK3 also interfered with T-cell influx, and reduced CD44 levels. Deletion of JNK2 had no effect. Thus, neuronal c-Jun is needed in regeneration, but JNK phosphorylation of the N-terminus mostly appears to not be required for its function.     

Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor

Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.     

A new ethanolamine phosphate-containing variant of the O-antigen of Shigella flexneri type 4a

The O-specific polysaccharide (O-antigen) structure of a Shigella flexneri type 4a strain from the Dysentery Reference Laboratory (London, UK) was elucidated in 1978 and its characteristic feature was found to be α-d-glucosylation of GlcNAc at position 6, which defines O-factor IV. Our NMR spectroscopic studies of the O-specific polysaccharides of two other strains belonging to S. flexneri type 4a (G1668 from Adelaide, Australia, and 1359 from Moscow, Russia) confirmed the carbohydrate backbone structure but revealed in both strains an additional component, ethanolamine phosphate (EtnP), attached at position 3 of one of the rhamnose residues:

Full-size image (5K)

Phosphorylation has not been hitherto reported in any S. flexneri O-antigen. Reinvestigation of the O-specific polysaccharide of S. flexneri type 4b showed that it is not phosphorylated and confirmed its structure established earlier.     

2'-O-methyl-modified anti-MDR1 fork-siRNA duplexes exhibiting high nuclease resistance and prolonged silencing activity

The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.     

Deposition of pectin/poly-L-lysine multilayers with pectins of varying degrees of esterification

The effect of pectin esterification on the assembly of multilayers consisting of poly-L-lysine (PLL) and pectin was studied using surface plasmon resonance (SPR), Fourier transform infrared-attenuated total reflection spectroscopy (FTIR-ATR), and a quartz crystal microbalance with dissipation monitoring (QCMD). With each layer deposited, there was a progressive increase in mass. The net charge of the multilayers was positive and increased with increasing degree of esterification of the pectin. Multilayer fabrication involved a limited fractionation of the pectin preparations, with the more highly esterified pectins having a weaker affinity for PLL. The multilayers were relatively hydrated structures with estimates of solids content in the range 10-32% w/w. The more highly esterified pectins had a tendency to form more hydrated structures, which showed a strong deswelling when PLL was added to a freshly deposited pectin layer.     

HyBMP5-8b, a BMP5-8 orthologue, acts during axial patterning and tentacle formation in hydra

Developmental gradients play a central role in axial patterning in hydra. As part of the effort towards elucidating the molecular basis of these gradients as well as investigating the evolution of the mechanisms underlying axial patterning, genes encoding signaling molecules are under investigation. We report the isolation and characterization of HyBMP5-8b, a BMP5-8 orthologue, from hydra. Processes governing axial patterning are continuously active in adult hydra. Expression patterns of HyBMP5-8b in normal animals and during bud formation, hydra's asexual form of reproduction, were examined. These patterns, coupled with changes in patterns of expression in manipulated tissues during head regeneration, foot regeneration as well as under conditions that alter the positional value gradient indicate that the gene is active in two different processes. The gene plays a role in tentacle formation and in patterning the lower end of the body axis.     

Potential role of transforming growth factor beta1 in drug resistance of tumor cells

Acquired drug resistance of tumor cells is frequently observed in cancer patients undergoing chemotherapy. We studied murine leukemia L1210 cells sensitive and resistant to the cytotoxic action of cisplatin and showed that cisplatin-resistant leukemia cells were also refractory to TGF beta1-dependent growth inhibition and apoptosis. Addressing the question about the mechanisms responsible for the cross-resistance to cisplatin and TGF beta1, we found that cisplatin- and TGF beta1-resistant L1210 cells possessed a decreased expression of type I TGF beta1 receptor, while the expression of type II TGF beta1 receptor was not affected. Western blot analysis of Smad proteins 2, 3, 4, 6, and 7, which participate in signal transduction pathway down-stream of the TGF beta1 receptors, revealed an increased expression of Smad 6, inhibiting TGF beta1 action, only in cisplatin- and TGF beta1-resistant L1210 cells. TGF beta1 and especially the cytotoxic mistletoe agglutinin increased Smad 6 expression in TGF beta1-sensitive but not in TGF beta1-resistant L1210 cells. TGF beta1-resistant L1210 cells also differed from TGF beta1-sensitive cells by the lack of expression of the pro-apoptotic p53 protein and higher level of expression of the anti-apoptotic Bcl-2 protein. Thus, the described co-expression of tumor cell refractoriness to an anti-cancer drug and to the inhibitory cytokine TGF beta1 is accompanied by multiple changes in the TGF beta1 signal transduction pathway and in other regulatory systems of the target cells. Besides, we found that various anti-tumor drugs and cytotoxic plant lectins increased the level of TGF beta1 expression in both TGFbeta1-sensitive and -resistant L1210 cells. A hypothesis is proposed that TGFbeta1 can at least partly mediate the effect of cell-stressing agents and, thus, the development of TGF beta1 resistance may be responsible for the appearance of tumor cell refractoriness to the action of some anti-cancer drugs.