Pivaloylcodeine,a new codeine derivative,for the inhibition of morphine glucuronidation. An in vitro study in the rat

We have previously found that phenanthrenic opioids, including codeine, modulate morphine glucuronidation in the rat. Here codeine and five of its derivatives were compared in their effects on the synthesis of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) from morphine by rat liver microsomal preparations, and by primary cultures of rat hepatocytes previously incubated for 72 h with either codeine or its derivatives. Acetylcodeine and pivaloylcodeine shared the capability of the parent compound of inhibiting the synthesis of M3G by liver microsomes through a noncompetitive mechanism of action. Their IC₅₀ were 3.25, 2.27, and 4.32 μM, respectively. Dihydrocodeine, acetyldihydrocodeine, and lauroylcodeine were ineffective. In all the experimental circumstances M6G was undetectable in the incubation medium. In primary hepatocyte cultures codeine only inhibited M3G formation, but with a lower efficacy than that observed with microsomes (IC₅₀ 20.91 vs 4.32 μM). Preliminary results show that at micromolar concentrations codeine derivatives exhibit a low rate of affinity for μ opiate receptors. In conclusion, acetyl and pivaloyl derivatives of codeine noncompetitively inhibit liver glucuronidation of morphine interacting with microsomes. This study further strengths the notion that phenanthrenic opioids can modulate morphine glucuronidation independently from their effects on μ opiate receptors.     

Molecular cloning and characterization of two novel autophagy-related genes belonging to the ATG8 family from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Rhipicephalus (Boophilus) microplus is an obligate haematophagous arthropod and the major problem for cattle industry due to economic losses it causes. The parasite shows a remarkable adaptability to changing environmental conditions as well as an exceptional ability to survive long-term starvation. This ability has been related to a process of intracellular protein degradation called autophagy. This process in ticks is still poorly understood and only few autophagy-related (ATG) genes have been characterized. The aim of the present study was to examine the ESTs database, BmiGI, of R. microplus searching for ATG homologues. We predicted five putative ATG genes, ATG3, ATG4, ATG6 and two ATG8s. Further characterization led to the identification of RmATG8a and RmATG8b, homologues of GABARAP and MAP1LC3, respectively, and both of them belonging to the ATG8 family. PCR analyses showed that the expression level of RmATG8a and RmATG8b was higher in egg and larval stages when compared to ovary and midgut from adult ticks. This up-regulation coincides with the period in which ticks are in a starvation state, suggesting that autophagy is active in R. microplus.     

Network medicine: linking disorders

The molecular events underlying many human hereditary disorders remain to be discovered despite the significant advances made in molecular biology and genetics in the past years. Given the complexity of cellular systems and the interplay between different functional modules, it is becoming increasingly evident that profound insights into human disease cannot be derived by analyzing single genetic defects. The generation of different types of disease interaction networks has recently emerged as a unifying approach that holds the promise of shedding some light on common pathological mechanisms by placing the single disorders into a larger context. In this review, I summarize the rationale behind these disease networks and different ways of constructing them. Finally, I highlight some of the first results that have been obtained by systematically analyzing the intertwined relationships between human disorders because they suggest that the current disease classification does not always sufficiently reflect biologically and medically relevant disease relationships.     

Exposure to 50 Hz electromagnetic field raises the levels of the anti-apoptotic protein BAG3 in melanoma cells

The expression of the anti‐apoptotic protein BAG3 is induced in several cell types by exposure to high temperature, oxidants, and other stressful agents. We investigated whether exposure to 50 Hz electromagnetic fields raised BAG3 levels in the human melanoma cell line M14, in vitro and in orthotopic xenografts. Exposure of cultured cells or xenografts for 6 h or 4 weeks, respectively, produced a significant (P < 0.01) increase in BAG3 protein amounts. Interestingly, at the same times, we could not detect any significant variation in the levels of HSP70/72 protein or cell apoptosis. These results confirm the stressful effect of exposure to ELF in human cells, by identifying BAG3 protein as a marker of ELF‐induced stress. Furthermore, they suggest that BAG3 induction by ELF may contribute to melanoma cell survival and/or resistance to therapy. J. Cell. Physiol. 226: 2901–2907, 2011. © 2011 Wiley‐Liss, Inc.     

The strawberry plant defense mechanism: a molecular review

Strawberry, a small fruit crop of great importance throughout the world, has been considered a model plant system for Rosaceae, and is susceptible to a large variety of phytopathogenic organisms. Most components and mechanisms of the strawberry defense network remain poorly understood. However, from current knowledge, it seems clear that the ability of a strawberry plant to respond efficiently to pathogens relies first on the physiological status of injured tissue (pre-formed mechanisms of defense) and secondly on the general ability to recognize and identify the invaders by surface plant receptors, followed by a broad range of induced mechanisms, which include cell wall reinforcement, production of reactive oxygen species, phytoalexin generation and pathogenesis-related protein accumulation. Dissection of these physiological responses at a molecular level will provide valuable information to improve future breeding strategies for new strawberry varieties and to engineer strawberry plants for durable and broad-spectrum disease resistance. In turn, this will lead to a reduction in use of chemicals and in environmental risks. Advances in the understanding of the molecular interplay between plant (mainly those considered model systems) and various classes of microbial pathogens have been made in the last two decades. However, major progress in the genetics and molecular biology of strawberry is still needed to uncover fully the way in which this elaborate plant innate immune system works. These fundamental insights will provide a conceptual framework for rational human intervention through new strawberry research approaches. In this review, we will provide a comprehensive overview and discuss recent advances in molecular research on strawberry defense mechanisms against pathogens.     

Crystallographic evidence for active-site dynamics in the hydrolytic aldehyde dehydrogenases. Implications for the deacylation step of the catalyzed reaction

The overall chemical mechanism of the reaction catalyzed by the hydrolytic aldehyde dehydrogenases (ALDHs) involves three main steps: (1) nucleophilic attack of the thiol group of the catalytic cysteine on the carbonyl carbon of the aldehyde substrate; (2) hydride transfer from the tetrahedral thiohemiacetal intermediate to the pyridine ring of NAD(P)(+); and (3) hydrolysis of the resulting thioester intermediate (deacylation). Crystal structures of different ALDHs from several organisms-determined in the absence and presence of bound NAD(P)(+), NAD(P)H, aldehydes, or acid products-showed specific details at the atomic level about the catalytic residues involved in each of the catalytic steps. These structures also showed the conformational flexibility of the nicotinamide half of the cofactor, and of the catalytic cysteinyl and glutamyl residues, the latter being the general base that activates the hydrolytic water molecule in the deacylation step. The architecture of the ALDH active site allows for this conformational flexibility, which, undoubtedly, is crucial for catalysis in these enzymes. Focusing in the deacylation step of the ALDH-catalyzed reaction, here we review and systematize the crystallographic evidence of the structural features responsible for the conformational flexibility of the catalytic glutamyl residue, and for the positioning of the hydrolytic water molecule inside the ALDH active site. Based on the analysis of the available crystallographic data and of energy-minimized models of the thioester reaction intermediate, as well as on the results of theoretical calculations of the pK(a) of the carboxyl group of the catalytic glutamic acid in its three different conformations, we discuss the role that the conformational flexibility of this residue plays in the activation of the hydrolytic water. We also propose a critical participation in the water activation process of the peptide bond to which the catalytic glutamic acid in the intermediate conformation is hydrogen bonded.     

Paraptosis‐like cell death in Wistar rat granulosa cells

Follicular atresia, a common process present in all mammals, involves apoptotic and autophagic cell death. However, the participation of paraptosis, a type of caspase‐independent cell death, during follicular atresia is unknown. This study found swollen endoplasmic reticulum in the granulosa cells of adult Wistar rats. Calnexin was used as a marker of the endoplasmic reticulum at the ultrastructural and optical levels. The cells with swelling of the endoplasmic reticulum were negative to the TUNEL assay and active caspase‐3 immunodetection, indicating that this swelling is not part of any apoptotic or autophagic process. Additionally, immunodetection of the CHOP protein was used as a marker of endoplasmic reticulum stress, and this confirmed the presence of the paraptosis process. These data suggest that paraptosis‐like cell death is associated with the death of granulosa cells during follicular atresia in adult Wistar rats.     

Protective Effect of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> on DMH-Induced Colon Carcinogenesis in Mice

The administration of probiotics is a promising approach to reduce the prevalence of colon cancer, a multifactorial disease, with hereditary factors, as well as environmental lifestyle-related risk factors. Biogenic polyamines, putrescine, spermidine, and spermine are small cationic molecules with great roles in cell proliferation and differentiation as well as regulation of gene expression. Ornithine decarboxylase is the first rate-limiting enzyme for polyamine synthesis, and upregulation of ornithine decarboxylase activity and polyamine metabolism has been associated with abnormal cell proliferation. This paper is focused on studying the protective role of Lactobacillus casei ATCC 393 in a chemically induced mouse model of colon carcinogenesis, directing our attention on aberrant crypt foci as preneoplastic markers, and on polyamine metabolism as a possible key player in carcinogenesis. BALB/c mice were administered 1,2-dimethylhydrazine dihydrochloride (DMH) to induce colon cancer (20 mg/kg body weight, subcutaneous, twice a week for 24 weeks). L. casei ATCC 393 was given orally (10⁶ CFU, twice a week), 2 weeks before DMH administration. Hematoxylin and eosin staining, high-performance liquid chromatography, and Western blotting were used to evaluate aberrant crypt foci, urinary polyamines, and ornithine decarboxylase expression in the colon. The experimental data showed that the preventive administration of L. casei ATCC 393 may delay the onset of cancer as it significantly reduced the number of DMH-induced aberrant crypt foci, the levels of putrescine, and the expression of ornithine decarboxylase. Hence, this probiotic strain has a prospective role in protection against colon carcinogenesis, and its antimutagenic activity may be associated with the maintenance of polyamine metabolism.