SIRT1 is involved in adrenocortical cancer growth and motility

Adrenocortical cancer (ACC) is a rare tumour with unfavourable prognosis, lacking an effective treatment. This tumour is characterized by IGF-II (insulin-like growth factor II) overproduction, aromatase and ERα (oestrogen receptor alpha) up-regulation. Previous reports suggest that ERα expression can be regulated by sirt1 (sirtuin 1), a nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylases that modulates activity of several substrates involved in cellular stress, metabolism, proliferation, senescence, protein degradation and apoptosis. Nevertheless, sirt1 can act as a tumour suppressor or oncogenic protein. In this study, we found that in H295R and SW13 cell lines, sirt1 expression is inhibited by sirtinol, a potent inhibitor of sirt1 activity. In addition, sirtinol is able to decrease ACC cell proliferation, colony and spheroids formation and to activate the intrinsic apoptotic mechanism. Particularly, we observed that sirtinol interferes with E2/ERα and IGF1R (insulin growth factor 1 receptor) pathways by decreasing receptors expression. Sirt1 involvement was confirmed by using a specific sirt1 siRNA. More importantly, we observed that sirtinol can synergize with mitotane, a selective adrenolitic drug, in inhibiting adrenocortical cancer cell growth. Collectively, our data reveal an oncogenic role for sirt1 in ACC and its targeting could implement treatment options for this type of cancer.     

Interaction of cobalt(II) bovine carbonic anhydrase with pyridine-2,6-dicarboxylate ion and other chelating ligands

The removal of cobalt from cobalt(II) bovine carbonic anhydrase by pyridine-2-carboxylate, pyridine-2,6-dicarboxylate and 5-methyl-1,10-phenanthroline occurs via formation of an intermediate. This is presumed to be a ternary adduct of cobalt(II) enzyme with the ligand. In this, metal-protein bonds are loosened, probably via distortion of the normal geometry, resulting in accelerated breakdown of the adduct to apoprotein, compared with the behavior of the cobalt(II) enzyme alone. With 2-carboxy-1,10-phenanthroline, removal of metal is very rapid but no adduct is observed. Values of stability constants of the adducts and rate constants for their decomposition to apoprotein and their formation from apoprotein and cobalt(II) complex were measured at pH 5.5 and 25°C. Formation and dissociation rate constants for the adduct of cobalt carbonic anhydrase with pyridine-2,6-dicarboxylate could be measured from pH 5 to 7 and 10° to 25°C by stopped flow. Values of thermodynamic parameters for the various reactions agreed well with those estimated from the kinetic data.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

Effects of neighbouring vegetation on eucalypt seedlings at two sites subject to different levels of abiotic stress

Abstract In spite of numerous studies on the effect of nutrient levels and/or standing crop on the intensity of resource competition the debate has not been resolved. Field studies that have used natural productivity gradients have generally supported the argument that competitive intensity and resource availability are positively correlated, whereas studies that have used artificial resource gradients have generally refuted the same argument. Here we report the results from study in which both approaches were used within the same system. We studied two species of eucalypt that occupy contrasting parts of the same landscape: Eucalyptus camaldulensis, found mostly along creek lines and in valleys with deep alluvial soils, and Eucalyptus microcarpa, found on hillsides and ridges with shallow soils. We studied the response of seedlings of the two species to the combined effects of competition and manipulated nutrient levels in a glasshouse experiment, and also investigated their responses to removal of neighbouring plants in the field. Eucalyptus microcarpa was less responsive to increased resource availability, which is consistent with one of the principal assumptions of Grime’s C‐S‐R model. In the glasshouse experiment both species of eucalypt responded in a qualitatively similar fashion to the combined effects of resource availability and competition: release from competition resulted in increased growth, but only in pots that received additional resources. In the field we found that neighbouring vegetation could severely limit the establishment of E. camaldulensis but the removal of neighbouring vegetation did not affect the performance of E. microcarpa seedlings. Eucalyptus camaldulensis seedlings suffered high levels of damage from herbivores. Our results thus generally support the predications of the C‐S‐R model, however, they indicate that the effects of competition and herbivory may be heavily confounded.     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

Characterization of experimental phenylketonuria augmentation of hyperphenylalaninemia with α-methylphenylalanine and p-chlorophenylalanine

Phenylalanine in conjuction with p-chlorophenylalanine or α-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterous effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing α-methylphenylalalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to expereimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, γ-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in t he brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age results obtained with animal models and the clinical findings in untreated phenylketonuric patients.     

Excitatory Amino Acids: Studies on the Biochemical and Chemical Stability of Ibotenic Acid and Related Compounds

The complex pharmacological profile (excitation/inhibition) of ibotenic acid on single neurons in the mammalian CNS prompted studies on the stability of ibotenic acid and a number of structurally related excitatory amino acids under different in vitro conditions in the presence or absence of enzymes. Ibotenic acid, (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and (RS)-alpha-amino-3-hydroxy-4-bromo-5-isoxazolepropionic acid (4-Br-homoibotenic acid) were all inhibitors of (S)-glutamic acid decarboxylase (GAD) in mouse brain homogenates, but only ibotenic acid was shown to undergo decarboxylation during incubation with brain homogenates. The formation of the decarboxylated product, muscimol, which primarily occurred in a synaptosomal fraction, was dependent on the presence of pyridoxal-5-phosphate (PALP) and was inhibited by (S)-glutamic acid, 3-mercaptopropionic acid (3MPA), aminooxyacetic acid (AOAA), and allyglycine, suggesting that ibotenic acid is a substrate for GAD. The overall decomposition rate for ibotenic acid (8.7 nmol min-1 mg-1 of protein), which apparently embraces other reactions in addition to decarboxylation to muscimol, was higher than the rate of decarboxylation of (S)-glutamic acid (3.2 nmol min-1 mg-1 of protein). At pH 7.4 and 37 degrees C, but in the absence of enzymes, none of the excitatory amino acids under study underwent any detectable decomposition, whereas ibotenic acid and 7-HPCA, but not AMPA and 4-Br-homoibotenic acid, decomposed, partially by decarboxylation, at 100 degrees C in a pH-dependent manner. In the presence of liver homogenates, ibotenic acid was also shown to decompose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent vs. Non‐Covalent Inhibition: Tackling Drug Resistance in EGFR – A Thorough Dynamic Perspective

A persistent challenge in the treatment of non‐small cell lung cancer (NSCLC) with EGFR is the emergence of drug‐resistant caused by somatic mutations. The EGFR L858R/T790 M double mutant (EGFR^DM) was found to be the most alarming variant. Despite the development of a wide range of inhibitors, none of them could inhibit EGFR^DM effectively. Recently, 11h and 45a , have been found to be potent inhibitors against EGFR^DM through two distinctive mechanisms, non‐covalent and covalent binding, respectively. However, the structural and dynamic implications of the two modes of inhibitions remain unexplored. Herein, two molecular dynamics simulation protocols, coupled with free‐energy calculations, were applied to gain insight into the atomistic nature of each binding mode. The comparative analysis confirmed that there is a significant difference in the binding free energy between 11h and 45a (ΔΔG_bind=?21.17 kcal/mol). The main binding force that governs the binding of both inhibitors is vdW, with a higher contribution for 45a . Two residues ARG841 and THR854 were found to have curtailed role in the binding of 45a to EGFR^DM by stabilizing its flexible alcohol chain. The 45a binding to EGFR^DM induces structural rearrangement in the active site to allow easier accessibility of 45a to target residue CYS797. The findings of this work can substantially shed light on new strategies for developing novel classes of covalent and non‐covalent inhibitors with increased specificity and potency.