Amine adducts of porphinato carbonylruthenium(II): studies of conformational effects and dynamics

The ¹H chemical shifts for 4-methylpyridine, imidazole, 2,5-dimethylimidazole, 2-undecylimidazole, pyrazole, 3,5-dimethylpyrazole, benzylamine, and α-methylbenzylamine bound to carbonylruthenium mesoporphyrin-IX-dimethyl ester or carbonylruthenium tetra-p-isopropylphenylporphyrin have been determined. The upfield shifts induced in the bound-nitrogen base resonances have been shown to arise principally from the magnetic anisotropy of the porphinato ligand. On this basis, models for calculating magnetic anisotropy in porphyrin systems have been tested and then applied to the determination of adduct geometry. These studies show that steric bulk of substituents ortho to the nitrogen binding determine the stability of various modes and control conformation in ligands. Furthermore, the results suggest adduct geometry and ligand-to-ring plane distances are relatively independent of porphyrin. Correlations between base structure and adduct stability were developed by measuring relative dissociation constants and first-order rate constants for dissociation. These measurements show a direct correlation between thermodynamic and kinetic stability and an inverse correlation between stability and increased steric bulk ortho to the donor atom.     

Hypoxia-activated ligand HAL-1/13 is lupus autoantigen Ku80 and mediates lymphoid cell adhesion in vitro

Hypoxia is known toinduce extravasation of lymphocytes and leukocytes duringischemic injury and increase the metastatic potential ofmalignant lymphoid cells. We have recently identified a new adhesionmolecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates thehypoxia-induced increases in lymphocyte and neutrophil adhesion toendothelium and hypoxia-mediated invasion of endothelial cellmonolayers by tumor cells. In this report, we used expression cloningto identify this molecule as the lupus antigen and DNA-dependentprotein kinase-associated nuclear protein, Ku80. TheHAL-1/13-Ku80 antigen is present on the surface of leukemic and solidtumor cell lines, including T and B lymphomas, myeloid leukemias,neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells.Transfection and ectopic expression of HAL-1/13-Ku80 on (murine)NIH/3T3 fibroblasts confers the ability of these normally nonadhesivecells to bind to a variety of human lymphoid cell lines. This adhesioncan be specifically blocked by HAL-1/13 or Ku80-neutralizingantibodies. Loss of expression variants of these transfectantssimultaneously lost their adhesive properties toward human lymphoidcells. Hypoxic exposure of tumor cell lines resulted in upregulation ofHAL-1/13-Ku80 expression at the cell surface, mediated byredistribution of the antigen from the nucleus. These studies indicatethat the HAL-1/13-Ku80 molecule may mediate, in part, thehypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.

     

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.     

Long-Term Effects of Interprofessional Biopsychosocial Rehabilitation for Adults with Chronic Non-Specific Low Back Pain: A Multicentre,Quasi-Experimental Study

Background

Improvement of the long-term effectiveness of multidisciplinary ortho-paedic rehabilitation (MOR) in the management of chronic non-specific low back pain (CLBP) remains a central issue for health care in Germany. We developed an interprofessional and interdisciplinary, biopsychosocial rehabilitation concept named “PASTOR” to promote self-management in adults with CLBP and compared its effectiveness with the current model of MOR.

Methods

A multicentre quasi-experimental study with three measurement time points was implemented. 680 adults aged 18 to 65 with CLBP were assed for eligibil-ity in three inpatient rehabilitation centres in Germany. At first the effects of the MOR, with a total extent of 48 hours (control group), were assessed. Thereafter, PASTOR was implemented and evaluated in the same centres (intervention group). It consisted of six interprofessional modules, which were provided on 12 days in fixed groups, with a total extent of 48 hours. Participants were assessed with self-report measures at baseline, discharge, and 12 months for functional ability (primary outcome) using the Hannover Functional Ability Questionnaire (FFbH-R) and vari-ous secondary outcomes (e.g. pain, health status, physical activity, pain coping, pain-related cognitions).

Results

In total 536 participants were consecutively assigned to PASTOR (n=266) or MOR (n=270). At 12 months, complete data of 368 participants was available. The adjusted between-group difference in the FFbH-R at 12 months was 6.58 (95% CI 3.38 to 9.78) using complete data and 3.56 (95% CI 0.45 to 6.67) using available da-ta, corresponding to significant small-to-medium effect sizes of d=0.42 (p<0.001) and d=0.10 (p=0.025) in favour of PASTOR. Further improvements in secondary out-comes were also observed in favour of PASTOR.

Conclusion

The interprofessional and interdisciplinary, biopsychosocial rehabilita-tion program PASTOR shows some improvements of the long-term effectiveness of inpatient rehabilitation in the management of adults with CLBP. Further insights into mechanisms of action of complex intervention programs are required.

Trial Registration

ClinicalTrials.gov NCT02056951     

Protein kinase C delta is required for survival of cells expressing activated p21RAS

Inhibition of protein kinase C (PKC) activity in transformed cells and tumor cells containing activated p21(RAS) results in apoptosis. To investigate the pro-apoptotic pathway induced by the p21(RAS) oncoprotein, we first identified the specific PKC isozyme necessary to prevent apoptosis in the presence of activated p21(RAS). Dominant-negative mutants of PKC, short interfering RNA vectors, and PKC isozyme-specific chemical inhibitors directed against the PKCdelta isozyme demonstrated that PKCdelta plays a critical role in p21(RAS)-mediated apoptosis. An activating p21(RAS) mutation, or activation of the phosphatidylinositol 3-kinase (PI3K) Ras effector pathway, increased the levels of PKCdelta protein and activity in cells, whereas inhibition of p21(RAS) activity decreased the expression of the PKCdelta protein. Activation of the Akt survival pathway by oncogenic Ras required PKCdelta activity. Akt activity was dramatically decreased after PKCdelta suppression in cells containing activated p21(RAS). Conversely, constitutively activated Akt rescued cells from apoptosis induced by PKCdelta inhibition. Collectively, these findings demonstrate that p21(RAS), through its downstream effector PI3K, induces PKCdelta expression and that this increase in PKCdelta activity, acting through Akt, is required for cell survival. The p21(RAS) effector molecule responsible for the initiation of the apoptotic signal after suppression of PKCdelta activity was also determined to be PI3K. PI3K (p110(C)(AAX), where AA is aliphatic amino acid) was sufficient for induction of apoptosis after PKCdelta inhibition. Thus, the same p21(RAS) effector, PI3K, is responsible for delivering both a pro-apoptotic signal and a survival signal, the latter being mediated by PKCdelta and Akt. Selective suppression of PKCdelta activity and consequent induction of apoptosis is a potential strategy for targeting of tumor cells containing an activated p21(RAS).     

Terminal sialic acids on CD44 N‐glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains

Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein‐ligand binding involved in cell–cell and cell–matrix interactions. CD44 is a single‐pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N‐terminal extracellular hyaluronan‐binding domain (HABD); specifically, sialic acid capped N‐glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N‐glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously‐formed charge‐paired hydrogen bonding interactions between the negatively‐charged sialic acid residues and positively‐charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. Proteins 2014; 82:3079–3089. © 2014 Wiley Periodicals, Inc.     

Di‐ and tri‐nucleotide repeat microsatellites for the mealy plum aphid,Hyalopterus pruni

Hyalopterus pruni is an invasive aphid pest in California. To study the population biology of this pest both in California and its native Mediterranean region, we have developed 11 di‐ and tri‐nucleotide repeat microsatellite markers. Each locus amplified in individuals representing the full range of geographical regions and host plants where Hyalopterus is found. Polymorphism was high, ranging from six to 22 alleles per locus in the individuals screened. These loci represent the first microsatellites developed for Hyalopterus and they should be of great value in studying the invasion biology and population structure of this insect pest.