Identification of N,N-arylalkyl-picolinamide derivatives targeting the RNA-binding protein HuR, by combining biophysical fragment-screening and molecular hybridization.

Hu proteins are members of the RNA-binding protein (RBP) family and play a pivotal role in the regulation of post-transcriptional processes. Through interaction with selected mRNAs, RBPs regulate their function and stability; as a consequence, RBP dysregulation can cause abnormal translation of key proteins involved in several pathologies. In the past few years, this observation has sparked interest to develop new treatments against these pathologies by using small molecules able to modulate RBP activity. Among the four Hu proteins, we have directed our efforts towards the isoform HuR, which is mainly involved in cancer, inflammation and retinopathy. Aimed at developing compounds able to modulate the stability of HuR-mRNA complexes, in the present work, we applied a biophysical fragment screening by assessing a library of halogen-enriched heterocyclic fragments (HEFLibs) via Surface Plasmon Resonance (SPR) and Saturation Transfer Difference (STD) NMR to select promising fragments able to interact with HuR. One selected fragment and a few commercially available congeners were exploited to design and synthesize focused analogues of compound N-(3-chlorobenzyl)-N-(3,5-dihydroxyphenethyl)-4-hydroxybenzamide (1), our previously reported hit. STDNMR spectroscopy, molecular modeling, and SPR offered further insight into the HuR-small molecule interaction and showed that fragment-based approaches represent a promising and yet underexplored strategy to tackle such unusual targets. Lastly, fluorescence polarization (FP) studies revealed the capability of the new compounds to interfere with the formation of the HuR-mRNA complex. This is, to our knowledge, the first fragment-based campaign performed on the Hu protein class, and one of the few examples in the larger RBP field and constitutes an important step in the quest for the rational modulation of RBPs and related RNA functions by small molecules.

Protein kinase C activation affects, via the mRNA-binding Hu-antigen R/ELAV protein, vascular endothelial growth factor expression in a pericytic/endothelial coculture model.

PURPOSE: To explore whether, following direct contact, there is mutual influence between pericytes (PC) and endothelial cells (EC), and to establish whether protein kinase C (PKC) activation, a condition associated with hyperglycemia, can affect, via the mRNA-binding Hu-antigen R (HuR)/ELAV protein, the expression of vascular endothelial growth factor (VEGF). METHODS: PC and EC were cultured separately or in direct contact (1:1 ratio), and exposed or not to phorbol esters, a PKC activator (100 nM for 15 min). Barrier integrity was evaluated by measuring endothelial electrical resistance and permeability to sodium fluorescein. Immunocytochemistry was performed to visualize EC and PC in coculture, and to evaluate phorbol 12-myristate-13-acetate (PMA)-induced HuR translocation. PKCßI/ßII, HuR, and VEGF protein content was measured with western blotting, VEGF secretion in cell culture medium was evaluated with enzyme-linked immunosorbent assay (ELISA), and quantification of VEGF mRNA was performed with real-time quantitative PCR. RESULTS: In monocultures, VEGF mRNA/protein basal levels were more elevated in PC than in EC. However, the basal expression of VEGF protein, but not mRNA, in PC and EC was affected by culture conditions. In fact, physical contact with PC upregulated VEGF protein levels in the EC, while VEGF was downregulated in PC cocultured with EC. In this last condition, PKCßII and HuR protein basal levels were also decreased in monocultured PC. Moreover, in basal conditions, the amount of VEGF released from the coculture was higher than from the monocultures. Direct activation of PKCß induced HuR translocation from the nuclear area to the cytoplasm, and increased the protein levels of the kinase itself, HuR, and VEGF in PC and EC in both culture conditions. Concerning VEGF mRNA, PKC activation induced an increase in PKC levels only in monocultured EC and, conversely, a significant decrease in the same transcript amount in cocultured PC. PMA stimulus also led to a significant increase in VEGF secretion in coculture. CONCLUSIONS: When cocultured with PC, EC form a significantly tighter barrier than the endothelial monolayer. The physical contact leads to opposite changes in VEGF protein levels in PC and EC. In particular, in basal conditions, cocultured PC seemed to downregulate their own expression of this proproliferating factor, as well as that of PKCßII and HuR, likely to maintain the 1:1 ratio with the cohabiting EC. In mono- and cocultured PC/EC, PKC direct activation led to a similar increase in PKCßI/ßII, HuR, and VEGF protein levels, changes that may also occur at early stages of diabetic retinopathy. The release of VEGF in the medium was favored by physical contact between PC and EC and was further increased by PMA exposure. In contrast with the effects on VEGF protein, PKCß activation induced modifications in VEGF mRNA content that are different in function of the cell type and the culture conditions. These findings suggest that the changes in the VEGF protein and transcript observed in PC/EC can be ascribed to distinct and concomitant pathways. Further studies on this in vitro coculture model would be useful to better understand the PC/EC interaction in physiologic and pathological conditions.

The PKCbeta/HuR/VEGF pathway in diabetic retinopathy.

We investigated whether the diabetes-related PKCbeta activation affects VEGF expression through the mRNA-stabilizing human embryonic lethal abnormal vision (ELAV) protein, HuR, in the retina of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection. Retinal tissues were processed to detect PKCbetaI, PKCbetaII, VEGF and HuR contents, as well as HuR phosphorylation. Immunoprecipitation coupled to RT-PCR was employed to evaluate HuR binding to VEGF mRNA in RiboNucleoProteic (RNP) complexes. Statistical analysis was performed by ANOVA followed by an appropriate post hoc comparison test. Following experimental diabetes PKCbetaI and PKCbetaII levels were increased compared to sham; there was also a PKC-mediated phosphorylation/activation of HuR. These effects were blunted by the in vivo co-administration of a selective PKCbeta inhibitor. A specific binding between the HuR protein and the VEGF mRNA was also detected. The PKCbeta/HuR activation was accompanied by enhanced VEGF protein expression that was, again, blunted by the PKCbeta inhibitor. These findings first demonstrate the activation, in the retina, of the PKCbeta/HuR/VEGF pathway following experimental diabetes and disclose a new potential pharmacological target to counteract pathologies implicating VEGF deregulation, such as diabetic retinopathy.

Senescence of the brain: focus on cognitive kinases.

Ageing is characterized by alterations in brain anatomy and physiology, finally contributing to an impairment in cognitive functions, such as memory. The most relevant observations indicate that senescent-related cognitive decline is not only due to neuronal loss, instead, functional changes occurring over time play a key role. Overall, these modifications are indeed responsible for an altered interneuronal communication that can represent, rather than morphological modifications, the primum movens leading to cognitive decline. Among the age-induced changes underlying alterations in neuronal communication and synaptic plasticity, those related to neurotransmitter/neurotrophin systems and downstream signalling pathways are of great relevance. In particular, considering that protein kinases play a strategic role aimed to convert the extracellular signals into biological responses, functional alterations on kinases may directly contribute to age-dependent neuronal dysfunctions. Within this context, numerous studies point out on several kinases as positive regulators for memory function and suggest that various memory disturbances are the result of a deficit in kinase signalling pathways. Many kinases associated with synaptic function are indeed age-sensitive; in fact, various studies in senescent animals indicate that a reduction in kinases expression/function in some brain areas correlates with ageing and memory decline. In line with these concepts, pharmacological modulation of kinases may lead to neuroprotective effects that can prevent or counteract age-related memory impairment. This review will mainly focus on the age-induced changes on Protein Kinase C (PKC), Protein Kinase A (PKA), Calcium/calmodulin-dependent Protein Kinase (CaMK), Tyrosine Kinase, widely accepted as key actors in signalling pathways associated with memory.

Involvement of Ca2+-dependent PKCs in the adaptive changes of mu-opioid pathways to sympathetic denervation in the guinea pig colon.

In the guinea pig colon, chronic sympathetic denervation entails supersensitivity to inhibitory mu-opioid agents modulating cholinergic neurons. The mechanism underlying such adaptive change has not yet been unravelled, although protein kinase C (PKC) may be involved. A previous study indirectly demonstrated that activation of mu-opioid receptors on myenteric neurons facilitates PKC activity. Such coupling may counteract the inhibitory action of mu-opioid agents on acetylcholine overflow, since PKC, per se, increases this parameter. After chronic sympathetic denervation such restraint abates, representing a possible mechanism for development of supersensitivity to mu-opioid agents. In the present study, this hypothesis was further investigated. After chronic sympathetic denervation, Ca(2+)-dependent PKC activity was reduced in colonic myenteric plexus synaptosomes. The mu-opioid agent, DAMGO, increased Ca(2+)-dependent PKC activity in synaptosomes obtained from normal, but not from denervated animals. In myenteric synaptosomes obtained from this experimental group, protein levels of Ca(2+)-dependent PKC isoforms betaI, betaII and gamma decreased, whereas alpha levels increased. In whole-mount preparations, the four Ca(2+)-dependent PKC isoforms co-localized with mu-opioid receptors on subpopulations of colonic myenteric neurons. The percentage of neurons staining for PKCbetaII, as well as the number of mu-opioid receptor-positive neurons staining for PKCbetaII, decreased in denervated preparations. The same parameters related to PKCalpha, betaI or gamma remained unchanged. Overall, the present data strengthen the concept that mu-opioid receptors located on myenteric neurons are coupled to Ca(2+)-dependent PKCs. After chronic sympathetic denervation, a reduced efficiency of this coupling may predominantly involve PKCbetaII, although also PKCbetaI and gamma, but not PKCalpha, may be implicated.

Post-transcriptional regulation of HSP70 expression following oxidative stress in SH-SY5Y cells: the potential involvement of the RNA-binding protein HuR.

Brain aging is associated with a progressive imbalance between intracellular concentration of Reactive Oxygen Species (ROS) and cells ability to activate defensive genes. Heat Shock Protein 70 (HSP70) has been shown to act as a fundamental defensive mechanism for neurons exposed to an oxidant challenge, and its expression decreases during senescence. In the present report we show that the RNA-binding protein ELAV/HuR can affect, post-transcriptionally, the fate of HSP70 mRNA following H(2)O(2)-mediated oxidative stress in SH-SY5Y human neuroblastoma cells. As a consequence of H(2)O(2) treatment (1mM for 30 minutes), HSP70 mRNA accumulates in the ribosomes associated to the cytoskeleton, where parallel Western blotting experiments reveal statistically significant increase for both HuR and HSP70 protein levels. We also confirm the capability of HuR to bind to HSP70 mRNA, and describe how the biological effect of this ELAV protein on the HSP70 mRNA could be due to a direct phosphorylation in serine/threonine residues of HuR itself by the early (10 minutes) H(2)O(2)-mediated activation of PKC alpha. Our findings shed light on the post-transcriptional regulation of HSP70 expression, suggesting the existence of a new molecular cascade -involving PKC/HuR/HSP70- that possibly represents an early event in the cellular response to H(2)O(2)-mediated oxidative stress in SH-SY5Y human neuroblastoma cells. The present results lead us to speculate that an impairment in this regulatory mechanism might directly contribute to the defective cellular response to oxidative stress, thus helping to dissect a potential tool useful to counteract some aspects associated to cerebral senescence.

Defining a neuron: neuronal ELAV proteins.

Neuronal cells strongly depend on the control exerted by RNA-binding proteins (RBPs) on gene expression for the establishment and maintenance of their phenotype. Neuronal ELAV (nELAV) proteins are RBPs able to influence virtually every aspect of the postsynthesis fate of bound mRNAs, from polyadenylation, alternative splicing and nuclear export to cytoplasmic localization, stability and translation. They enhance gene expression through the last two, best documented activities, increasing mRNA half-life and promoting protein synthesis by a still-unknown molecular mechanism. Developmentally, nELAV proteins have been shown to act as inducers of the transition between neural stem/progenitor cells and differentiation-committed cells, also assisting these neuroblasts in the completion of their maturation program. In brain physiology, they are also the first RBPs demonstrated to have a pivotal role in memory, where they probably control mRNA availability for translation in subcellular domains, thereby providing a biochemical means for selective increase in synaptic strength.

An epidemic of dengue 3 in far north Queensland, 1997-1999.

OBJECTIVES: To describe an epidemic of dengue type 3 that occurred in far north Queensland in 1997-1999 and its influence on the further development of dengue prevention and control strategies. DESIGN: Epidemiological and laboratory investigation of cases, entomological surveys and phylogenetic analysis of dengue virus isolates. MAIN OUTCOME MEASURES: Numbers and characteristics of confirmed cases; Breteau Index (BI; number of containers breeding Aedes aegypti per 100 premises); effect of control measures on mosquito populations; genetic homology of epidemic virus with other dengue virus isolates. RESULTS: The epidemic lasted 70 weeks and comprised 498 confirmed cases in three towns (Cairns, Port Douglas and Mossman); 101 patients (20%) were admitted to hospital. Median interval between symptom onset and notification was seven days (range, 0-53 days), and cumulative duration of viraemia of public health significance was 2,072 days. BIs in affected areas were high, particularly in Mossman (45) and Port Douglas (31). Control measures significantly reduced mosquito populations (assessed as number of ovitraps containing Ae. aegypti eggs and mean number of eggs per trap [P< 0.05 for both]). However, transmission persisted in several foci, in part due to undetected waterfilled containers breeding Ae. aegypti. The epidemic virus belonged to serotype 3; phylogenetic analysis suggested it was imported from Thailand. CONCLUSIONS: The epidemic had greater morbidity than other recent Queensland epidemics of dengue and was harder to control, necessitating substantial revision of the Dengue Fever Management Plan for North Queensland. The epidemic's severity supports the hypothesis that dengue viruses from South East Asia are more virulent than others.

[Cancer of the appendix. A report of 2 cases]. / Il ca. dell'appendice. Osservazione di due casi.

The paper reports two cases of adenocarcinoma of the appendix. Following a review of the literature on this neoplasia, it concludes that prognosis is poor on account of the early spread of the disease, the low ratio of suspected disease, and difficulties of diagnosis prior to surgery. The preferred surgical treatment is right hemicolectomy which consents a 5-year survival rate of 50% in comparison to 20% achieved with appendectomy alone. The authors also stress the importance of carrying out histological tests after any type of appendectomy.

[Unusual cause of post-traumatic hemoperitoneum: rupture of enterogenous cysts]. / Insolita causa di emoperitoneo post-traumatico: rottura di cisti enterogena.

Authors present a rare complication of multiple enteric cystes: hemoperitoneum. A little girl 4 years old was operated on by suspected acute appendicitis. The surgery showed hemoperitoneum by bleeding from broken (post-traumatic) paradigiunal cyst. Other 5 cystes were present near the first one. Authors review shortly some titles of bibliography about duplication of the alimentary tract and hemoperitoneum.

[Acute torsion of the greater omentum (presentation of 4 clinical cases and bibliographical notes)]. / La torsione acute del grande omento (presentazione di 4 casi clinici e note bibliografiche).

Authors present 4 cases of acute torsion of the greater omentum (2 cases primary, 2 cases secondary. They analyze a short review of the literature and point of the rarity of this disease that, mimicking an acute appendicitis, make difficult right diagnosis before surgery.

[Acute lithiasic cholecystitis, situs viscerum inversus, vena porta preduodenale (considerations on a clinical case)]. / Colecistite acuta litiasica, situs viscerum inversus, vena porta preduodenale (considerazioni circa un caso clinico).

Authors report a case of preduodenal portal vein in a woman 68 years old with acute cholecystitis in situs viscerum inversus adbominis and levocardia. Surgery was difficult. They shortly analyze also etiology and clinical manifestations of situs inversus and PPV, with related malformations. They conclude that it is important to recognize the presence of situs inversus preoperatively and everytime suspect other anatomical anomalies.