Mdmx and Mdm2: Brothers in Arms?

The p53 tumor suppressor pathway is inactivated in most if not all human tumors. In about 50% of the cases this is accomplished directly by gene mutations. The tumors that retain wild type p53 frequently show defects either in effector target genes, or in the expression of p53 regulatory proteins. The Mdm2 protein is generally considered THE master regulator of the p53 tumor suppressor activity. Recently, however, the Mdm2-related protein Mdmx is taking the stage in the p53-Mdm2-Mdmx play. We summarize here observations unambiguously assigning a critical role for the Mdmx protein in the regulation of p53 function during development and tumor formation.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

5′-Phosphonates of Ribonucleosides and 2′-Deoxyribonucleosides: Synthesis and Antiviral Activity

Abstract

5′-Phosphonates of natural 2′-deoxynucleosides and ribonucleosides were synthesized by condensation of 3′-O-acylated 2′-deoxynucleosides or 2′,3′-substituted (2′,3′-O-isopropylidene, 2′,3′-O-methoxymethylene or 2′,3′-O-ethoxymethylene) ribonucleosides. As condensing agents, either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulphonyl chloride were used. Nucleoside 5′-ethoxycarbonylphosphonates were converted into corresponding nucleoside 5′-aminocarbonylphosphonates by action of ammonia in methanol or aqueous ammonia. 5′-Hydrogenphosphonothioates of thymidine and 3′-deoxythymidine were obtained by reaction of phosphinic acid in the presence of pivaloyl chloride with 3′-O-acetylthymidine or 3′-deoxythymidine, respectively, followed by addition of powedered sulfur. 5′-O-methylenephosphonates of thymidine and 2′-deoxyadenosine were prepared by intramolecular reaction of corresponding 3′-O-iodomethylphosphonates under basic conditions. All compounds were tested for inhibition of several viruses, including HSV-2 and CMV, but showed no activity. A few compounds insignificantly inhibited HIV-1 reproduction. Thymidine 5′-hydrogenphosphonate neutralized anti-HIV action of 3′-azido-3′-deoxythymidine (AZT) and it indirectly showed that even some nucleoside 5′-phosphonates could be partly hydrolyzed in cell culture to corresponding nucleosides.

5′-Phosphonates of modified 2′-deoxynucleosides in which one group in a phosphate residue is substituted for hydrogen, alkyl or other groups, have shown to be potent biologically     

2-Pyridylmetallocenes: Part I. Electrophilic halogenation of 2-pyridylferrocene. Molecular structures of 2-pyridylferrocene and its α-brominated and -fluorinated derivatives. Synthesis of 2-pyridylruthenocene and 2-pyridylcymantrene

A new high-yield synthesis of 2-pyridylferrocene (1) without formation of the 1,1′-disubstituted product has been developed. Also the corresponding ruthenocene and cymantrene derivatives [C₅H₄(2-C₅H₄N)]ML_n (ML_n = Ru(C₅H₅) (2), Mn(CO)₃ (3)) were prepared and fully characterized. Ortho-lithiation of 1 followed by electrophilic halogenation yielded [C₅H₃X(2-C₅H₄N)]Fe(C₅H₅) [X = F (4), Cl (5), Br (6), I (7)], with 4 only being the second reported and first fully characterized fluoroferrocene. The molecular structures of 1, 4 and 6 have been determined by X-ray crystallography.     

N-Aminoacyl and N-hydroxyacyl derivatives of diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside: Synthesis,antimicrobial and hemolytic activities

Diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside is a semisynthetic saponin with antimicrobial and antitumor activities. To search for more effective analogues, N-aminoacyl and N-hydroxyacyl derivatives of this saponin were synthesized conventionally and with microwave assistance, and tested against the human pathogenic fungi and Gram-positive and Gram-negative bacteria. None of the tested compounds exhibit activity against Gram-negative bacteria. Almost all of the synthesized N-aminoacyl saponins exhibit antifungal activity and act effectively against Gram-positive bacteria, some better than the parent compound. The best acting saponins are the same size and possess sarcosine or l- or d-alanine attached to the parent glucosaminoside. Shorter and longer aminoacyl residues are less advantageous. d-Alanine derivative is the most effective against Gram positive bacteria. Structure-activity relationship (SAR) analysis indicates that the free α-amino group in aminoacyl residue is necessary for antimicrobial activities of the tested saponins. (N-Acetyl)aminoacyl and N-hydroxyacyl analogs are inactive. Measurements of the hemolytic activities demonstrate that the best acting saponins are not toxic towards human red blood cells.     

Penam Sulfones and β-Lactamase Inhibition: SA2-13 and the Importance of the C2 Side Chain Length and Composition

β-Lactamases are the major reason β-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, β-lactamase inhibitors are currently being developed. Presently, there are only three that are in clinical use (clavulanate, sulbactam and tazobactam). In order to address this important medical need, we explored a new inhibition strategy that takes advantage of a long-lived inhibitory trans-enamine intermediate. SA2-13 was previously synthesized and shown to have a lower k _react than tazobactam. We investigated here the importance of the carboxyl linker length and composition by synthesizing three analogs of SA2-13 (PSR-4-157, PSR-4-155, and PSR-3-226). All SA2-13 analogs yielded higher turnover numbers and k _react compared to SA2-13. We next demonstrated using protein crystallography that increasing the linker length by one carbon allowed for better capture of a trans-enamine intermediate; in contrast, this trans-enamine intermediate did not occur when the C2 linker length was decreased by one carbon. If the linker was altered by both shortening it and changing the carboxyl moiety into a neutral amide moiety, the stable trans-enamine intermediate in wt SHV-1 did not form; this intermediate could only be observed when a deacylation deficient E166A variant was studied. We subsequently studied SA2-13 against a relatively recently discovered inhibitor-resistant (IR) variant of SHV-1, SHV K234R. Despite the alteration in the mechanism of resistance due to the K→R change in this variant, SA2-13 was effective at inhibiting this IR enzyme and formed a trans-enamine inhibitory intermediate similar to the intermediate seen in the wt SHV-1 structure. Taken together, our data reveals that the C2 side chain linker length and composition profoundly affect the formation of the trans-enamine intermediate of penam sulfones. We also show that the design of SA2-13 derivatives offers promise against IR SHV β-lactamases that possess the K234R substitution.     

C2 and C2C12 murine skeletal myoblast models of atrophic and hypertrophic potential: Relevance to disease and ageing?

Reduced muscle mass and increased susceptibility to TNF‐induced degradation accompany inflamed ageing and chronic diseases. Furthermore, C₂ myoblasts display diminished differentiation and increased susceptibility to TNF‐α‐induced cell death versus subcloned C₂C₁₂ cells, providing relevant models to assess: differentiation (creatine kinase), growth (protein), death (trypan‐blue) and anabolic/catabolic parameters (RT‐PCR) over 72 h ± TNF‐α (20 ng ml^?1). At 48 and 72 h, respectively, larger myotubes and significantly higher CK activity (320.26 ± 6.82 vs. 30.71 ± 2.5, P < 0.05; 544.94 ± 27.7 vs. 39.4 ± 3.37 mU mg ml^?1, P < 0.05), fold increases in myoD (21.45 ± 3.12 vs. 3.97 ± 1.76, P < 0.05; 31.07 ± 3.1 vs. 6.82 ± 1.93, P < 0.05) and myogenin mRNA (241.8 ± 40 vs. 36.80 ± 19.3, P < 0.05; 440 ± 100.5 vs. 201.1 ± 86, P < 0.05) were detected in C₂C₁₂ versus C₂. C₂C₁₂ showed significant increases in IGF‐I mRNA (243.05 ± 3.87 vs. 105.75 ± 21.95, P < 0.05), reduced proliferation and significantly lower protein expression (1.21 ± 0.28 vs. 1.79 ± 0.29 mg ml^?1, P < 0.05) at 72 h versus C₂ cells. Significant temporal reductions in C₂C₁₂ IGFBP2 mRNA (28.02 ± 15.44, 13.82 ± 8.07, 6.92 ± 4.37, P < 0.05) contrasted increases in C₂s (4.31 ± 3.31, 13.02 ± 9.92, 82.9 ± 58.9, P < 0.05) at 0, 48 and 72 h, respectively. TNF‐α increased cell death in C₂s (2.67 ± 1.54%, 34.42 ± 5.39%, 29.71 ± 5.79% (0, 48, 72 h), P < 0.05), yet was without effect in C₂C₁₂s at 48 h but caused a small significant increase at 72 h (9.88 ± 4.02% (TNF‐α) vs. 6.17 ± 0.749% (DM), 72 h). TNF‐α and TNFRI mRNA were unchanged; however, larger reductions in IGF‐I (8.2‐ and 7.5‐fold vs. 4.5‐ and 4.1‐fold (48, 72 h)), IGF‐IR (2‐fold vs. no‐significant reduction (72 h)) and IGFBP5 (3.24 vs. 1.38 (48 h) and 2.21 vs. 1.71 (72 h), P < 0.05) mRNA were observed in C₂ versus C₂C₁₂ with TNF‐α. This investigation provides insight into regulators of altered basal hypertrophy and TNF‐induced atrophy, providing a model for future investigation into therapeutic initiatives for ageing/wasting disorders. J. Cell. Physiol. 225: 240–250, 2010. © 2010 Wiley‐Liss, Inc.     

Part 2: Design, synthesis and evaluation of hydroxyproline-derived α2δ ligands

Conformational constraint has been used to design a potent series of α₂δ ligands derived from the readily available starting material (2S,4R)-hydroxy-l-proline. The ligands have improved physicochemistry and potency compared to their linear counterparts (described in our earlier publication) and the lead compound has been progressed to clinical development.     

2′-Deoxy-2′-C-trifluoromethyl β-D-Ribonucleoside Analogues: Synthesis and Antiviral Evaluations

Abstract

Hitherto unknown 2′-deoxy-2′-C-trifluoromethyl-β-D-ribonucleoside derivatives bearing the five naturally occurring nucleic acid bases have been synthesized. The compounds were tested for their activity against HIV, HBV and several RNA viruses, but they did not show significant antiviral effect.     

IGFBP-2 and ?5: important regulators of normal and neoplastic mammary gland physiology

The insulin-like growth factor (IGF) axis plays an important role in mammary gland physiology. In addition, dysregulation of this molecular axis may have a causal role in the aetiology and development of breast cancer (BC). This report discusses the IGF axis in normal and neoplastic mammary gland with special reference to IGF binding proteins (IGFBPs) -2 and −5. We describe how these high affinity binders of IGF-1 and IGF-2 may regulate local actions of growth factors in an autocrine and/or paracrine manner and how they also have IGF-independent effects in mammary gland. We discuss clinical studies which investigate both the prognostic value of IGFBP-2 and −5 expression in BC and possible involvement of these genes in the development of resistance to adjuvant endocrine therapies.     

Structural and functional determinants of protein kinase CK2��: facts and open questions

Ser/Thr protein kinase CK2 is involved in several fundamental processes that regulate the cell life, such as cell cycle progression, gene expression, cell growth, and differentiation and embryogenesis. In various cancers, CK2 shows a markedly elevated activity that has been associated with conditions that favor the onset of the tumor phenotype. This prompts to numerous studies aimed at the identification of compounds that are able to inhibit the catalytic activity of this oncogenic kinase, in particular, of ATP-competitive inhibitors. The many available crystal structures indicate that this enzyme owns some regions of remarkable flexibility which were associated to important functional properties. Of particular relevance is the flexibility, unique among protein kinases, of the hinge region and the following helix αD. This study attempts to unveil the structural bases of this characteristic of CK2. We also analyze some controversial issues concerning the functional interpretation of structural data on maize and human CK2 and try to recognize what is reasonably established and what is still unclear about this enzyme. This analysis can be useful also to outline some principles at the basis of the development of effective ATP-competitive CK2 inhibitors.     

Phenylbenzenesulfonates and -sulfonamides as 17β-hydroxysteroid dehydrogenase type 2 inhibitors: Synthesis and SAR-analysis

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) converts the potent estrogen estradiol into the weakly active keto form estrone. Because of its expression in bone, inhibition of 17β-HSD2 provides an attractive strategy for the treatment of osteoporosis, a condition that is often caused by a decrease of the active sex steroids. Currently, there are no drugs on the market targeting 17β-HSD2, but in multiple studies, synthesis and biological evaluation of promising 17β-HSD2 inhibitors have been reported. Our previous work led to the identification of phenylbenzenesulfonamides and -sulfonates as new 17β-HSD2 inhibitors by ligand-based pharmacophore modeling and virtual screening. In this study, new molecules representing this scaffold were synthesized and tested in vitro for their 17β-HSD2 activity to derive more profound structure-activity relationship rules.     

p34cdc2: the S and M kinase?

In the yeast cell cycle, the induction of two very different processes, DNA synthesis (S-phase) and mitosis (M-phase), requires the same serine/threonine-specific protein kinase p34cdc2, which has been highly conserved through evolution. On the basis of work conducted largely in multicellular eukaryotes, it has recently been suggested that p34cdc2 is able to perform these two mutually exclusive roles by phosphorylating different sets of substrates through a cell cycle-dependent association with other proteins that dictate the substrate specificity of the protein kinase. To recognize its mitotic substrates, p34cdc2 associates with one of the cyclins--a family of proteins of two distinct but related types (A and B) characterized by their periodic destruction at each mitosis. In interphase, the formation of a complex between p34cdc2 and another protein (or proteins) would allow the phosphorylation of a different set of proteins involved in the G1 to S transition. This review focuses on the evidence for this appealing simple model and the nature of the putative substrates proposed.     

Stu2p and XMAP215: turncoat microtubule-associated proteins?

The dynamics of microtubule-based (MT) cytoskeletons are controlled by a variety of accessory proteins: microtubule-associated proteins (MAPs), which usually stabilize MTs, and microtubule-destabilizers. Two related MAPs, XMAP215 and Stu2p, are known to stabilize MTs. However, recent studies report that these proteins have a MT-destabilizing function as well. Here we discuss the implications of these reports.     

Light- and CO2-saturated photosynthesis: enhancement by oxygen

Oxygen may enhance CO₂-saturated photosynthesis in intact leaves, which display the Warburg effect when illuminated at the current atmospheric level of CO₂ and O₂, of about 350 μl l⁻¹ and 21%, respectively. The magnitude of the stimulation depends on irradiance. The K _M(O₂) of the stimulation is 128 μM (10.6% O₂). Maximum enhancement in wheat leaves is 6.1 and 5.3 μmol m⁻² s⁻¹ under 27.9 and 18.7 mW cm⁻², respectively, corresponding to a 25–30% increase in the ribulose 1,5-bisphosphate (RuBP) turnover rate if compared with O₂-free ambient gas phase. The stimulation appears in 5–10 s after a sharp increase in O₂. In response to a decrease in O₂, the new stabilized rate is reached in 5–7 min. The stimulation does not involve any increase in the activity of Rubisco. The effect correlates with increased concentration of RuBP. Oxygen enhances CO₂-saturated photosynthesis by acting as a terminal electron acceptor in the photosynthetic electron transport. The magnitude of the effect may be adopted as an index of the pseudocyclic photophosphorylation in vivo.     

Prostaglandin E2 and T cells: friends or foes?

Our understanding of the key players involved in the differential regulation of T-cell responses during inflammation, infection and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. With respect to this, the inhibitory role of the lipid mediator prostaglandin E(2) (PGE(2)) in T-cell immunity has been documented since the 1970s. Studies that ensued investigating the underlying mechanisms substantiated the suppressive function of micromolar concentrations of PGE(2) in T-cell activation, proliferation, differentiation and migration. However, the past decade has seen a revolution in this perspective, since nanomolar concentrations of PGE(2) have been shown to potentiate Th1 and Th17 responses and aid in T-cell proliferation. The understanding of concentration-specific effects of PGE(2) in other cell types, the development of mice deficient in each subtype of the PGE(2) receptors (EP receptors) and the delineation of signalling pathways mediated by the EP receptors have enhanced our understanding of PGE(2) as an immune-stimulator. PGE(2) regulates a multitude of functions in T-cell activation and differentiation and these effects vary depending on the micro-environment of the cell, maturation and activation state of the cell, type of EP receptor involved, local concentration of PGE(2) and whether it is a homeostatic or inflammatory scenario. In this review, we compartmentalize the various aspects of this complex relationship of PGE(2) with T lymphocytes. Given the importance of this molecule in T-cell activation, we also address the possibility of using EP receptor antagonism as a potential therapeutic approach for some immune disorders.     

Bioactive eicosanoids: Role of prostaglandin F2α and F2-isoprostanes in inflammation and oxidative stress related pathology

Oxidative stress and inflammation are supposed to be the key players of several acute and chronic diseases, and also for progressive aging process. Eicosanoids, especially prostaglandin F_2α (PGF_2α) and F₂-isoprostanes are endogenous compounds that are involved both in physiology and the above mentioned pathologies. These compounds are biosynthesized mainly from esterified arachidonic acid through both enzymatic and non-enzymatic free radical-catalysed reactions in vivo, respectively. They have shown to possess potent biological activities in addition to their application as biomarkers of oxidative stress and inflammation. Recent advancement of methodologies has made it possible to quantify these compounds more reliably and apply them in various in vivo studies successfully. Today, experimental and clinical studies have revealed that both PGF_2α and F₂-isoprostanes are involved in severe acute or chronic inflammatory conditions such as rheumatic diseases, asthma, risk factors of atherosclerosis, diabetes, ischemia-reperfusion, septic shock and many others. These evidences promote that assessment of bioactive PGF_2α and F₂-isoprostanes simultaneously in body fluids offers unique non-invasive analytical opportunity to study the function of these eicosanoids in physiology, oxidative stress-related and inflammatory diseases, and also in the determination of potency of various radical scavengers, anti-inflammatory compounds, drugs, antioxidants and diet.