Residues in the alternative reading frame tumor suppressor that influence its stability and p53-independent activities

The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARF's anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala¹⁴ and Thr³¹) were found to destabilize the protein while two others (Val²⁴ and Ala⁴¹) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significant biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val²⁴ was required for p53-independent growth suppression whereas multiple residues (Val²⁴, Thr³¹, Ala⁴¹ and His⁶⁰) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability.     

Evolution: the paradox of sperm leviathans

Sexual selection theory predicts that sperm competition will push males to produce more, smaller sperm. Paradoxically, in the fruitfly Drosophila bifurca sperm competition is rife but males produce few, giant sperm--the largest known. A recent study reconciles the evolution of giant sperm with theory.     

Spare interactions of highly potent [Arg14,Lys15]nociceptin for cooperative induction of ORL1 receptor activation

[Arg¹⁴,Lys¹⁵]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg¹⁴,Lys¹⁵]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg¹⁴,Lys¹⁵]. Trp208Ala was found to bind strongly both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.     

NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1

EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated ¹⁵N, ¹³C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded β sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor α4β1. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of Long-Term Feeding with Acetyl-L-Carnitine on the Age-Related Changes in Rat Brain Lipid Composition: A Study by 31P NMR Spectroscopy

Changes in brain lipid composition have been determined in 24 months-old Fischer rats with respect to 6 months-old ones. The cerebral levels of sphingomyelin and cholesterol were found to be significantly increased in aged rats, whereas the amount of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid appear to be unaffected by aging. Long-term feeding with acetyl-L-carnitine was able to reduce the age-dependent increase of both sphingomyelin and cholesterol cerebral levels with no effect on the other measured phospholipids. These findings shown that changes in membrane lipid metabolism and/or composition represent one of the alterations occurring in rat brain with aging, and that long-term feeding with acetyl-L-carnitine can be useful in normalizing these age-dependent disturbances.     

Differential Sensitivity of Basal and Opioid-Stimulated Low Km GTPase to Guanine Nucleotide Analogs

In membranes derived from NG108-15 cells, the opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5'-O-(2-thio)diphosphate (GDP beta S), guanosine 5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] and guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) inhibit the basal enzymatic activity with the order of potency GTP gamma S greater than Gpp (NH)p greater than GDP beta S. In the presence of DADLE, the inhibition isotherms of GDP beta S and Gpp(NH)p are shifted to the right five- and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTP gamma S for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTP gamma S produce a concentration-dependent increase in the Km(app) of GTPase, without affecting its Vmax, indicating a competitive inhibition. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibition. Both Gpp(NH)p and GTP gamma S, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase. These findings indicate that the basal and the opioid-stimulated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs.     

Sodium Bicarbonate Treatment during Transient or Sustained Lactic Acidemia in Normoxic and Normotensive Rats

Introduction

Lactic acidosis is a frequent cause of poor outcome in the intensive care settings. We set up an experimental model of lactic acid infusion in normoxic and normotensive rats to investigate the systemic effects of lactic acidemia per se without the confounding factor of an underlying organic cause of acidosis.

Methodology

Sprague Dawley rats underwent a primed endovenous infusion of L(+) lactic acid during general anesthesia. Normoxic and normotensive animals were then randomized to the following study groups (n = 8 per group): S) sustained infusion of lactic acid, S+B) sustained infusion+sodium bicarbonate, T) transient infusion, T+B transient infusion+sodium bicarbonate. Hemodynamic, respiratory and acid-base parameters were measured over time. Lactate pharmacokinetics and muscle phosphofructokinase enzyme''s activity were also measured.

Principal Findings

Following lactic acid infusion blood lactate rose (P<0.05), pH (P<0.05) and strong ion difference (P<0.05) drop. Some rats developed hemodynamic instability during the primed infusion of lactic acid. In the normoxic and normotensive animals bicarbonate treatment normalized pH during sustained infusion of lactic acid (from 7.22±0.02 to 7.36±0.04, P<0.05) while overshoot to alkalemic values when the infusion was transient (from 7.24±0.01 to 7.53±0.03, P<0.05). When acid load was interrupted bicarbonate infusion affected lactate wash-out kinetics (P<0.05) so that blood lactate was higher (2.9±1 mmol/l vs. 1.0±0.2, P<0.05, group T vs. T+B respectively). The activity of phosphofructokinase enzyme was correlated with blood pH (R2 = 0.475, P<0.05).

Conclusions

pH decreased with acid infusion and rose with bicarbonate administration but the effects of bicarbonate infusion on pH differed under a persistent or transient acid load. Alkalization affected the rate of lactate disposal during the transient acid load.     

On the generation of OH<Superscript>·</Superscript> radical species from H<Subscript>2</Subscript>O<Subscript>2</Subscript> by Cu(I) amyloid beta peptide model complexes: a DFT investigation

According to different studies, the interaction between amyloid β-peptide (Aβ) and copper ions could yield radical oxygen species production, in particular the highly toxic hydroxyl radical OH^· that is suspected to contribute to Alzheimer’s disease pathogenesis. Despite intensive experimental and computational studies, the nature of the interaction between copper and Aβ peptide, as well as the redox reactivity of the system, are still matter of debate. It was proposed that in Cu(II) → Cu(I) reduction the complex Cu(II)–Aβ could follow a multi-step conformational change with redox active intermediates that may be responsible for OH^· radical production from H₂O₂ through a Fenton-like process. The purpose of this work is to evaluate, using ab initio Density Functional Theory computations, the reactivity of different Cu(I)–Aβ coordination modes proposed in the literature, in terms of OH^· production. For each coordination model, we considered the corresponding H₂O₂ adduct and performed a potential energy surface scan along the reaction coordinate of O–O bond dissociation of the peroxide, resulting in the production of OH^· radical, obtaining reaction profiles for the evaluation of the energetic of the process. This procedure allowed us to confirm the hypothesis according to which the most populated Cu(I)–Aβ two-histidine coordination is not able to perform efficiently H₂O₂ reduction, while a less populated three-coordinated form would be responsible for the OH^· production. We show that coordination modes featuring a third nitrogen containing electron-donor ligand (an imidazole ring of an histidine residue is slightly favored over the N-terminal amine group) are more active towards H₂O₂ reduction.