Abdominal magnetic resonance imaging in small rodents using a clinical 1.5 T MR scanner

Because of superior soft-tissue contrast compared to other imaging techniques, non-invasive abdominal magnetic resonance imaging (MRI) is ideal for monitoring organ regeneration, tissue repair, cancer stage, and treatment effects in a wide variety of experimental animal models. Currently, sophisticated MR protocols, including technically demanding procedures for motion artefact compensation, achieve an MRI resolution limit of < 100 microm under ideal conditions. However, such a high spatial resolution is not required for most experimental rodent studies. This article describes both a detailed imaging protocol for MR data acquisition in a ubiquitously and commercially available 1.5 T MR unit and 3-dimensional volumetry of organs, tissue components, or tumors. Future developments in MR technology will allow in vivo investigation of physiological and pathological processes at the cellular and even the molecular levels. Experimental MRI is crucial for non-invasive monitoring of a broad range of biological processes and will further our general understanding of physiology and disease.     

Diorganotin(IV) complexes of dipeptides containing the alpha-aminoisobutyryl residue (Aib): preparation, structural characterization, antibacterial and antiproliferative activities of [(n-Bu)2 Sn(H(-1)L)] (LH=H-Aib-L-Leu-OH, H-Aib-L-Ala-OH)

Two new organotin(IV) complexes with dianionic dipeptides containing the α-aminoisobutyryl residue (Aib) as ligands are described. The solid complexes [(n-Bu)₂Sn(H₋₁L_A)] · 2MeOH (1 · 2MeOH) (L_AH = H-Aib-L-Leu-OH) and [(n-Bu)₂Sn(H₋₁L_B)] · MeOH (2 · MeOH) (L_BH = H-Aib-L-Ala-OH) have been isolated and characterized by single-crystal X-ray crystallography and spectroscopic techniques (H₋₁L²⁻ is the dianionic form of the corresponding dipeptide). Complexes 1 · 2MeOH and 2 · MeOH are monomeric with similar molecular structures. The doubly deprotonated dipeptide behaves as a N(amino), N(peptide), O(carboxylate) ligand and binds to the Sn^IV atom. The five-coordinate metal ion has a distorted trigonal bipyramidal geometry. A different network of intermolecular hydrogen bonds in each compound results in very dissimilar supramolecular features. The IR, far-IR, Raman and ¹¹⁹Sn NMR data are discussed in terms of the nature of bonding and known structures. The antibacterial and antiproliferative activities as well as the effect of the new compounds on pDNA were examined. Complexes 1 and 2 are active against the gram-positive bacteria Bacillus subtilis and Bacillus cereus. The IC₅₀ values reveal that the two compounds express promising cytotoxic activity in vitro against a series of cell lines.     

A genome-wide survey of human thioredoxin and glutaredoxin family pseudogenes

The thioredoxin/glutaredoxin family consists of small heat-stable proteins that have a highly conserved CXXC active site and that participate in the regulation of many redox reactions. We have searched the human genome sequence to find putative pseudogenes (non-functional copies of protein-coding genes) for all known members of this family. This survey has resulted in the identification of seven processed pseudogenes for human Trx1 and two more for human Grx1. No evidence for the presence of processed pseudogenes has been found for the remaining members of this family. In addition, we have been unable to detect any non-processed pseudogenes derived from any member of the family in the human genome. The seven thioredoxin pseudogenes can be divided into two groups: Trx1-psi2, -psi3, -psi4, -psi5 and -psi6 arose from the functional ancestor, whereas Trx1-psi1 and -psi7 originated from Trx1-psi2 and -psi6, respectively. In all cases, the pseudogenes originated after the human/rodent radiation as shown by phylogenetic analysis. This is also the case for Grx1-psi1 and Grx1-psi2, which are placed between rodent and human sequences in the phylogenetic tree. Our study provides a molecular record of the recent evolution of these two genes in the hominid lineage.     

Human mitochondrial thioredoxin. Involvement in mitochondrial membrane potential and cell death

Thioredoxins (Trx) are a class of small multifunctional redox-active proteins found in all organisms. Recently, we reported the cloning of a mitochondrial thioredoxin, Trx2, from rat heart. To investigate the biological role of Trx2 we have isolated the human homologue, hTrx2, and generated HEK-293 cells overexpressing Trx2 (HEK-Trx2). Here, we show that HEK-Trx2 cells are more resistant toward etoposide. In addition, HEK-Trx2 are more sensitive toward rotenone, an inhibitor of complex I of the respiratory chain. Finally, overexpression of Trx2 confers an increase in mitochondrial membrane potential, DeltaPsi(m). Treatment with oligomycin could both reverse the effect of rotenone and decrease the membrane potential suggesting that Trx2 interferes with the activity of ATP synthase. Taken together, these results suggest that Trx2 interacts with specific components of the mitochondrial respiratory chain and plays an important role in the regulation of the mitochondrial membrane potential.     

Insulin-Like Growth Factor 1 Receptor (IGF1R) Expression and Survival in Operable Squamous-Cell Laryngeal Cancer

Introduction

Prognosis of patients with operable laryngeal cancer is highly variable and therefore potent prognostic biomarkers are warranted. The insulin-like growth factor receptor (IGFR) signaling pathway plays a critical role in laryngeal carcinogenesis and progression.

Patients and Methods

We identified all patients with localized TNM stage I–III laryngeal cancer managed with potentially curative surgery between 1985 and 2008. Immunohistochemical (IHC) expression of IGF1R-alpha, IGF1R-beta and IGF2R was evaluated using the immunoreactive score (IRS) and mRNA levels of important effectors of the IGFR pathway were assessed, including IGF1R, IGF-binding protein 3 (IGFBP3), suppressor of cytokine signaling 2 (SOCS2) and members of the MAP-kinase (MAP2K1, MAPK9) and phosphatidyl-inositol-3 kinase (PIK3CA, PIK3R1) families. Cox-regression models were applied to assess the predictive value of biomarkers on disease-free survival (DFS) and overall survival (OS).

Results

Among 289 eligible patients, 95.2% were current or ex smokers, 75.4% were alcohol abusers, 15.6% had node-positive disease and 32.2% had received post-operative irradiation. After a median follow-up of 74.5 months, median DFS was 94.5 months and median OS was 106.3 months. Using the median IRS as the pre-defined cut-off, patients whose tumors had increased IGF1R-alpha cytoplasm or membrane expression experienced marginally shorter DFS and significantly shorter OS compared to those whose tumors had low IGF1R-alpha expression (91.1 vs 106.2 months, p = 0.0538 and 100.3 vs 118.6 months, p = 0.0157, respectively). Increased mRNA levels of MAPK9 were associated with prolonged DFS (p = 0.0655) and OS (p = 0.0344). In multivariate analysis, IGF1R-alpha overexpression was associated with a 46.6% increase in the probability for relapse (p = 0.0374). Independent predictors for poor OS included node-positive disease (HR = 2.569, p<0.0001), subglottic/transglottic localization (HR = 1.756, p = 0.0438) and IGF1R-alpha protein overexpression (HR = 1.475, p = 0.0504).

Conclusion

IGF1R-alpha protein overexpression may serve as an independent predictor of relapse and survival in operable laryngeal cancer. Prospective evaluation of the IGF1R-alpha prognostic utility is warranted.     

The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1.     

Modulators of the hedgehog signaling pathway

Since its discovery by C. Nüsslein-Volhard and E. F. Wieschaus, hedgehog (hh) signaling has come a long way. Today it is regarded as a key regulator in embryogenesis where it governs processes like cell proliferation, differentiation, and tissue patterning. Furthermore, in adults it is involved in the maintenance of stem cells, and in tissue repair and regeneration. But hh signaling has a second—much darker—face: it plays an important role in several types of human cancers where it promotes growth and enables proliferation of tumor stem cells. The etiology of medulloblastoma and basal cell carcinoma is tightly linked to aberrant hh activity, but also cancers of the prostate, the pancreas, the colon, the breasts, rhabdomyosarcoma, and leukemia, are dependent on irregular hh activity. Recent clinical studies have shown that hh signaling can be the basis of an important new class of therapeutic agents with far-reaching implications in oncology. Thus, modulation of hh signaling by means of small molecules has emerged as a valuable tool in combating these hh-dependent cancers. Cyclopamine, a unique natural product with a fascinating history, was the first identified inhibitor of hh signaling and its story is closely linked to the progress in the whole field. In this review we will trace the story of cyclopamine, give an overview on the biological modes of hh signaling both in untransformed and malignant cells, and finally present potent modulators of the hh pathway—many of them already in clinical studies. For more than 30 years now the knowledge on hh signaling has grown steadily—an end to this development is far from being conceivable.     

The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress

The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.