Cytostatic/Cytotoxic Effects of 5‐Fluorouridine Nucleolipids on Colon,Hepatocellular, and Renal Carcinoma Cells: in vitro Identification of a Potential Cytotoxic Multi‐Anticancer Drug

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5‐fluorouracil (5‐FU; 1 ). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or at the 2′,3′‐O position, i.e., 3a, 3b, 4 – 7 , and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT‐29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5‐FU ( 1 ) and 5‐FUrd ( 2a ). After 48 h of incubation, four derivatives, 3a, 3b, 5 , and 7 , showed inhibitory effects on the survival of HT‐29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side‐effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5 , and 6 did not exhibit any effects on survival of THP‐1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a , and the above‐mentioned derivatives in HT‐29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 μM after 2‐h or 4‐h treatment, as well as after 6‐h incubation at 40 μM (p<0.05). Real‐time PCR revealed that 40‐μM derivative 5 showed a 1.8‐fold increase of the pro‐apoptotic caspase‐3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1 , resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5‐FUrd derivatives such as compound 5 are more effective than 5‐FU or 5‐FUrd concerning a cytotoxic (vs. cytostatic (5‐FU, 5‐FUrd)) effect on different cancer cell lines, but without cytotoxic side‐effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi‐anti‐cancer drug.     

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats

Background  

Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.     

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[¹⁸F]fluoro-glucose (FDG) and 3′-deoxy-3′-[¹⁸F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC₅₀ values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 µM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.     

Influence of inverse dynamics methods on the calculation of inter-segmental moments in vertical jumping and weightlifting

Background  

A vast number of biomechanical studies have employed inverse dynamics methods to calculate inter-segmental moments during movement. Although all inverse dynamics methods are rooted in classical mechanics and thus theoretically the same, there exist a number of distinct computational methods. Recent research has demonstrated a key influence of the dynamics computation of the inverse dynamics method on the calculated moments, despite the theoretical equivalence of the methods. The purpose of this study was therefore to explore the influence of the choice of inverse dynamics on the calculation of inter-segmental moments.     

Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes

The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require gamma-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.     

Ensemble attribute profile clustering: discovering and characterizing groups of genes with similar patterns of biological features

Background  

Ensemble attribute profile clustering is a novel, text-based strategy for analyzing a user-defined list of genes and/or proteins. The strategy exploits annotation data present in gene-centered corpora and utilizes ideas from statistical information retrieval to discover and characterize properties shared by subsets of the list. The practical utility of this method is demonstrated by employing it in a retrospective study of two non-overlapping sets of genes defined by a published investigation as markers for normal human breast luminal epithelial cells and myoepithelial cells.     

Nucleoterpenes of Thymidine and 2′‐Deoxyinosine: Synthons for a Biomimetic Lipophilization of Oligonucleotides

2′‐Deoxyinosine ( 1 ) and thymidine ( 7 ) were N‐alkylated with geranyl and farnesyl moieties. These hydrophobic derivatives, 3a and 3b , and 9a and 9b , respectively, represent the first synthetic biomimetic nucleoterpenes and were subsequently 5′‐protected and converted into the corresponding 3′‐O‐phosphoramidites, 5a and 5b and 11a and 11b , respectively. The latter were used to prepare a series of lipophilized oligonucleotide dodecamers, a part of which were additionally labelled with indocarbocyanine fluorescent dyes (Cy3 or Cy5), 18 – 23 . The insertion of the lipooligonucleotides into, as well as duplex formation at artificial lipid bilayers was studied by single‐molecule fluorescence spectroscopy and fluorescence microscopy.     

Birth Origin Differentially Affects Depressive-Like Behaviours: Are Captive-Born Cynomolgus Monkeys More Vulnerable to Depression than Their Wild-Born Counterparts?

Background

Adverse early-life experience might lead to the expression of abnormal behaviours in animals and the predisposition to psychiatric disorder (e.g. major depressive disorder) in Humans. Common breeding processes employ weaning and housing conditions different from what happens in the wild.

Methods

The present study, therefore, investigated whether birth origin impacts the possible existence of spontaneous atypical/abnormal behaviours displayed by 40 captive-born and 40 wild-born socially-housed cynomolgus macaques in farming conditions using an unbiased ethological scan-sampling analysis followed by multifactorial correspondence and hierarchical clustering analyses.

Results

We identified 10 distinct profiles (groups A to J) that significantly differed on several behaviours, body postures, body orientations, distances between individuals and locations in the cage. Data suggest that 4 captive-born and 1 wild-born animals (groups G and J) present depressive-like symptoms, unnatural early life events thereby increasing the risk of developing pathological symptoms. General differences were also highlighted between the captive- and wild-born populations, implying the expression of differential coping mechanisms in response to the same captive environment.

Conclusions

Birth origin thus impacts the development of atypical ethologically-defined behavioural profiles, reminiscent of certain depressive-like symptoms. The use of unbiased behavioural observations might allow the identification of animal models of human mental/behavioural disorders and their most appropriate control groups.     

GTP Cyclohydrolase I Gene Polymorphisms Are Associated with Endothelial Dysfunction and Oxidative Stress in Patients with Type 2 Diabetes Mellitus

Background

The genetic background of atherosclerosis in type 2 diabetes mellitus (T2DM) is complex and poorly understood. Studying genetic components of intermediate phenotypes, such as endothelial dysfunction and oxidative stress, may aid in identifying novel genetic components for atherosclerosis in diabetic patients.

Methods

Five polymorphisms forming two haplotype blocks within the GTP cyclohydrolase 1 gene, encoding a rate limiting enzyme in tetrahydrobiopterin synthesis, were studied in the context of flow and nitroglycerin mediated dilation (FMD and NMD), intima-media thickness (IMT), and plasma concentrations of von Willebrand factor (vWF) and malondialdehyde (MDA).

Results

Rs841 was associated with FMD (p = 0.01), while polymorphisms Rs10483639, Rs841, Rs3783641 (which form a single haplotype) were associated with both MDA (p = 0.012, p = 0.0015 and p = 0.003, respectively) and vWF concentrations (p = 0.016, p = 0.03 and p = 0.045, respectively). In addition, polymorphism Rs8007267 was also associated with MDA (p = 0.006). Haplotype analysis confirmed the association of both haplotypes with studied variables.

Conclusions

Genetic variation of the GCH1 gene is associated with endothelial dysfunction and oxidative stress in T2DM patients.