Apo-10'-lycopenoic acid impacts adipose tissue biology via the retinoic acid receptors.

Apo-10'-lycopenoic acid (apo-10-lycac), a metabolite of lycopene, has been shown to possess potent biological activities, notably via the retinoic acid receptors (RAR). In the current study, its impact on adipose tissue and adipocytes was studied. In microarray experiments, the set of genes regulated by apo-10-lycac treatments was compared to the set of genes regulated by all-trans retinoic acid (ATRA), the natural ligand of RAR, in adipocytes. Approximately 27.5% of the genes regulated by apo-10-lycac treatments were also regulated by ATRA, suggesting a common ability in terms of gene expression modulation, possibly via RAR transactivation. The physiological impact of apo-10-lycac on adipose tissue biology was evaluated. If it had no effect on adipogenesis in the 3T3-L1 cell model, this metabolite may have a preventative effect against inflammation, by preventing the increase in the inflammatory markers, interleukin 6 and interleukin 1ß in various dedicated models. The ability of apo-10-lycac to transactivate the RAR and to modulate the transcription of RAR target gene was brought in vivo in adipose tissue. While apo-10-lycac was not detected in adipose tissue, a metabolite with a molecular weight with 2Da larger mass was detected, suggesting that a dihydro-apo-10'-lycopenoic acid, may be present in adipose tissue and that this compound could active or may lead to further active RAR-activating apo-10-lycac metabolites. Since apo-10-lycac treatments induce anti-inflammatory effects in adipose tissue but do not inhibit adipogenesis, we propose that apo-10-lycac treatments and its potential active metabolites in WAT may be considered for prevention strategies relevant for obesity-associated pathologies.

Macrophage activation, phagocytosis and intracellular calcium oscillations induced by scorpion toxins from Tityus serrulatus.

The research described here is focused upon studying the activation of mice peritoneal macrophages when submitted to in vitro effects of Tityus serrulatus scorpion venom and its major toxic peptides. Several functional events were analysed, such as: cytotoxicity, spreading, extent of phagocytosis, vacuole formation and changes of internal calcium concentration. Among the main results observed, when macrophages are subjected to the effects of soluble venom of Tityus serrulatus scorpion venom, a partially purified fraction (FII) or a pure toxin (Ts1), are an increment in the percentage of phagocytosis and vacuole formation, a decrement of the spreading ability, accompanied by oscillations of internal calcium concentration. The net results demonstrate that scorpion venom or its major toxins are effective stimulators of macrophage activity; the effect of whole soluble venom or partially purified fractions is due to the toxic peptides, seen here clearly with Ts1. The possible involvement of Na+-channels in these events is discussed. A basic understanding of the underlying molecular mechanisms responsible for macrophage activation should serve as a foundation for novel drug development aimed at modulating macrophage activity.

Neural responses to dynamic expressions of fear in schizophrenia.

Abnormalities in social functioning are a significant feature of schizophrenia. One critical aspect of these abnormalities is the difficulty these individuals have with the recognition of facial emotions, particularly negative expressions such as fear. The present work focuses on fear perception and its relationship to the paranoid symptoms of schizophrenia, specifically, how underlying limbic system structures (i.e. the amygdala) react when probed with dynamic fearful facial expressions. Seven paranoid and eight non-paranoid subjects (all males) with a diagnosis of schizophrenia took part in functional magnetic resonance imaging study (1.5T) examining neural responses to emerging fearful expressions contrasted with dissipating fearful expressions. Subjects viewed emerging and dissipating expressions while completing a gender discrimination task. Their brain activation was compared to that of 10 healthy male subjects. Increased hippocampal activation was seen in the non-paranoid group, while abnormalities in the bilateral amygdalae were observed only in the paranoid individuals. These patterns may represent trait-related hippocampal dysfunction, coupled with state (specifically paranoia) related amygdala abnormalities. The findings are discussed in light of models of paranoia in schizophrenia.

p57(Kip2) stabilizes the MyoD protein by inhibiting cyclin E-Cdk2 kinase activity in growing myoblasts.

We show that expression of p57(Kip2), a potent tight-binding inhibitor of several G(1) cyclin-cyclin-dependent kinase (Cdk) complexes, increases markedly during C2C12 myoblast differentiation. We examined the effect of p57(Kip2) on the activity of the transcription factor MyoD. In transient transfection assays, transcriptional transactivation of the mouse muscle creatine kinase promoter by MyoD was enhanced by the Cdk inhibitors. In addition, p57(Kip2), p21(Cip1), and p27(Kip1) but not p16(Ink4a) induced an increased level of MyoD protein, and we show that MyoD, an unstable nuclear protein, was stabilized by p57(Kip2). Forced expression of p57(Kip2) correlated with hypophosphorylation of MyoD in C2C12 myoblasts. A dominant-negative Cdk2 mutant arrested cells at the G(1) phase transition and induced hypophosphorylation of MyoD. Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk2 complexes was inhibited by p57(Kip2). In addition, the NH2 domain of p57(Kip2) necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulation in proliferative myoblasts. Taken together, our data suggest that repression of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57(Kip2) could play an important role in the accumulation of MyoD at the onset of myoblast differentiation.

The Drosophila melanogaster homologue of the Xeroderma pigmentosum D gene product is located in euchromatic regions and has a dynamic response to UV light-induced lesions in polytene chromosomes.

The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair. Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy. XPD has a 5'- to 3'-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation. We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD). DmXPD encodes a product that is highly related to its human homologue. The DmXPD protein is ubiquitous during development. In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic. At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei. Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs. UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage. This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Preventing long-lasting fear recovery using bilateral alternating sensory stimulation: A translational study.

Posttraumatic stress disorder (PTSD) is a highly debilitating and prevalent psychological disorder. It is characterized by highly distressing intrusive trauma memories that are partly explained by fear conditioning. Despite efficient therapeutic approaches, a subset of PTSD patients displays spontaneous recurrence of traumatic memories after successful treatment. The development of animal behavioral models mimicking the individual variability in treatment outcome for PTSD patients represent therefore an important challenge as it allows for the identification of predicting factors of resilience or susceptibility to relapse. However, to date, only few animal behavioral models of long-lasting fear recovery have been developed and their predictive validity has not been tested directly. The objectives of this study were twofold. First we aimed to develop a simple animal behavioral model of long-lasting fear recovery based on auditory cued fear conditioning and extinction learning, which recapitulates the heterogeneity of fear responses observed in PTSD patients after successful treatment. Second we aimed at testing the predictive validity of our behavioral model and used to this purpose a translational approach based (i) on the demonstration of the efficiency of Eye Movement Desensitization and Reprocessing (EMDR) therapy to reduce conditioned fear responses in PTSD patients and (ii) on the implementation in our behavioral model of an electrical bilateral alternating stimulation of the eyelid which mimics the core feature of EMDR. Our data indicate that electrical bilateral alternating stimulation of the eyelid during extinction learning alleviates long-lasting fear recovery of conditioned fear responses and dramatically reduces inter-individual variability. These results demonstrate the face and predictive validity of our animal behavioral model and provide an interesting tool to understand the neurobiological underpinnings of long-lasting fear recovery.

Dimerization of the amino terminal domain of p57Kip2 inhibits cyclin D1-cdk4 kinase activity.

Previous studies have led to the proposal that a single molecule of Cki can associate with the cyclin/Cdk complex to repress its activity. On the other hand, multiple inhibitor molecules are required to inhibit Cdks. In the present work, by using differently tagged p57Kip2 proteins we demonstrate that p57Kip2 can bind to itself in vitro and in vivo. Mutational deletion analysis showed that the NH2 terminal domain of p57Kip2 is necessary and sufficient to dimerization. Using an in vitro competition/association assay, we demonstrate that cyclin D1 alone, Cdk4 alone and/or cyclin D1/Cdk4 complexes do not compete for the p57Kip2 homodimers formation. However, a mutation in the alpha-helix domain of p57Kip2 (R33L) strongly reduced homodimer formation but did not modify interaction with cyclin D1-Cdk4 complexes. Also, increasing amounts of p57Kip2 lead in vivo to a significant augmentation in the level of p57Kip2 homodimerization associated with cyclin D1-Cdk4 complexes and to a marked inhibition of the cyclin D1-Cdk4 kinase activity. Altogether, these data suggest a model whereby p57Kip2 associates with itself by using the NH2 domain to form a homodimeric species which interacts with and inhibits the cyclin D1-Cdk4 complexes.

Ionic bases of the membrane potential and intracellular pH changes induced by speract in swollen sea urchin sperm.

Signal transduction initiated by the egg peptide, speract, in sea urchin sperm is not fully understood. Hypotonically swollen sperm are a suitable model to study peptide signal transduction. Ion substitution experiments now indicate (i) that the permeability to Na+, Ca2+, and Mg2+ contributes to the sperm resting membrane potential; (ii) the repolarization induced by nM concentrations of speract is Na+ dependent and mediated by an as yet unidentified channel; (iii) the depolarization triggered by nM concentrations of speract involves Ca2+ channels since it is Ca(2+)-dependent and blocked by Co2+ and Ni2+, two Ca2+ channel blockers; (iv) hyperpolarizing swollen sperm with valinomycin increases intracellular pH (pHi) in the same way as speract, thus the speract-induced hyperpolarization may be responsible for the pHi increase.

Serum pentosidine as an indicator of Alzheimer's disease.

Pentosidine, an advanced glycation end product (AGE), was assayed by HPLC in serum proteins from patients with Alzheimer type dementia (AD), patients with diabetes mellitus (D), and healthy (C) age-matched old subjects (mean age from each group = 84 years). Serum pentosidine was significantly different between the three groups despite similar renal function (serum creatinine < 160 micromol/L). In all groups of patients, pentosidine was independent of glycated hemoglobin (HbA1C) and the early glycation marker fructosamine and appeared to be an independent marker, mainly bound to serum albumin. Pentosidine could be an important factor useful for the diagnosis of Alzheimer's disease.

A new affinity adsorbent for the purification of phospholipases A1 and A2 from animal venoms.

Dimethyl-DL-2,3-distearoyloxy-propyl-2'-hydroxy-ethylammonium++ + (Rosenthal's inhibitor) was coupled to carboxyhexyl-Sepharose 4B, through carbodiimide chemistry. Phospholipase A2 from Heloderma horridum horridum and Crotalus adamanteus bind to the immobilized ligand in the presence of Ca2+ and can be easily eluted under acidic conditions or in the presence of a chelating agent, respectively. This affinity media proved to be effective also in the purification of a Ca2(+)-independent phospholipase A1 from vespid venom.

Synergy between ascorbate and alpha-tocopherol on fibroblasts in culture.

Ascorbate and tocopherol are important antioxidants that protect cells against oxidative stress. The interaction of ascorbate and alpha-tocopherol in cells is difficult to detect as both ascorbate and alpha-tocopherol are unstable in vitro in a biological medium. We examined the interactions between human dermal fibroblasts, ascorbate and alpha-tocopherol to determine the effects of the vitamins on growth and cell viability. The interaction of ascorbate and alpha-tocopherol was studied in a fibroblast culture medium during 48h. Ascorbate and alpha-tocopherol were detected by fluorimetry after high-performance liquid chromatography (HPLC). Cell growth and cell viability were studied by cell numeration after trypan blue staining. The ascorbate concentration fell in presence of alpha-tocopherol in cell culture medium under all experimental conditions, with or without cells. Ascorbate partly protected alpha-tocopherol but only in presence of cells. Cell viability was preserved by alpha-tocopherol whereas ascorbate enhanced fibroblast growth. The synergy between ascorbate and alpha-tocopherol corresponds to a consumption of ascorbate which spares alpha-tocopherol but only in presence of cells.

From the beginning: the basal transcription machinery and onset of transcription in the early animal embryo.

Transcription onset in the early animal embryo is a fundamental process required for proper embryonic development. Depending on the species, transcription onset occurs at what specifically appears to be different developmental stages. However, studies in early embryos from different animal models have shown that components of the basal transcription machinery play fundamental and highly regulated roles at the onset of transcription. The state of the basal transcription machinery in the embryo seems to be equivalent in different organisms at transcription onset. The dynamic balance between putative activators and repressors as well as the chromatin/cytoplasmic ratio seem to be coordinated with basal transcription factors in order to activate zygotic transcription. Here we discuss and compare the regulation of the basal transcription machineries and their activation in early embryos of different model organisms.

Molecular analysis and chromosome mapping of the H2A, H3 and H4 histone genes from the malaria vector Anopheles gambiae.

In this article we report the cloning and analysis of PCR generated fragments that encode H2A, H3 and H4 histone genes from the malaria vector An. gambiae. Sequence analysis indicated that some conservative changes are present in the An. gambiae H2A and H4 genes as compared with histone genes from other organisms. Divisional mapping showed that these genes map in division 20 on the left arm of the second chromosome. Southern blot experiments and the molecular characterization of the genomic fragment containing the H2A, H2B, H3 and H4 genes showed that they are organized in a cluster with an orientation different from the one found in other dipterans.

Cloning and characterization of cDNAs preferentially expressed in the ovary of the mosquito, Anopheles gambiae.

We used differential screening to isolate from an ovarian cDNA library two expressed sequences that are enriched substantially in ovaries of blood-fed female Anopheles gambiae, as compared to female carcass and male mosquitoes. One of these clones encodes an isoform of histone H2B, whose transcript is polyadenylated at the 3' end. The other cDNA clone encodes a protein that is highly conserved in evolution and has been implicated in growth control although its function is still obscure. Both genes can be used to study gene activation during An. gambiae oogenesis.

[Synthesis and properties of semisynthetic penicillins containing the furoxan nucleus]. / Sintesi e proprietà di penicilline semisintetiche contenenti il nucleo furossanico

Synthesis, antibacterial activity and stability in acid solution of potassium salts of 6-(3-methyl-4-furoxancarboxyamido)penicillanic acid and 6-(4-methyl-3-furoxancarboxyamido)penicillanic acid are reported.

Antisense suppression of the putative ribosomal protein S3A gene disrupts ovarian development in Drosophila melanogaster.

The Drosophila melanogaster homologue of the Anopheles gambiae C3 cDNA has been isolated and characterized by sequence analysis. The encoded protein was localized by immunochemical and immunocytochemical methods. The Drosophila C3 protein is highly similar to homologues of disputed function, which have previously been identified in fungi, plants and animals. The protein is ubiquitous and localized in the cytoplasm. Cell fractionation followed by detection with a specific antibody preparation shows that the protein is associated with the 40S ribosomal subunit. The C3 gene is located in section 101F of chromosome 4. Antisense transgenic analysis shows that this gene is essential for oogenesis. The most prominent phenotype resulting from antisense depletion of C3 RNA is disappearance of the follicular cells of the ovary (where the concentration of C3 protein is normally high) and abnormalities of the associated germline derivatives, leading to failure of egg production.

Changes in glycation of fibrous type I collagen during long-term in vitro incubation with glucose.

The course of glycation of calf skin fibrous type I collagen was monitored in vitro under physiological conditions during an 8-week incubation period in order to take into account the long half-life of this protein. The formation of glycated compounds was measured by determining fructosamine, pentosidine, and carboxymethyllysine content. The incubation conditions were as physiological as possible in sterile saline phosphate buffer, except glucose concentration. With incubation medium containing 200 mmol glucose, fibrous collagen underwent solubilization; in addition an increase in fructosamine, pentosidine, and carboxymethyllysine content in both solubilized and remaining insoluble collagen was noticed. There was a spontaneous, restricted, and time-dependent native glycated state of collagen; high concentration glucose enhanced the formation of glycated compounds and induced changes in solubility and glycoxidated products. The production of pentosidine during incubation without glucose should be considered as an event resulting from the initial fructosamine. Whereas the production of carboxymethyllysine during long-term incubation with glucose provided indirect proof of an additional oxidative process after early glycated product formation. These experimental observations provide insight into the in vivo context of advanced glycation end product formation in chronic hyperglycemia and aging.

In vitro glycoxidation of insoluble fibrous type I collagen: solubilization and advanced glycation end products.

The deleterious effects of glycoxidation are dependent on the half-life of proteins. Collagen, the main component of extracellular matrices, is a long live protein and thus may be sensitive to the glycoxidation process. We incubated calf skin fibrous type I collagen in PBS at 37 degrees C with glucose. The fibrous type I collagen was solubilized and an increase in the amount of advanced glycation end products of the solubilized fraction was observed. As there was no bacterial contamination and no proteolytic activities in the incubation medium, the solubilization of fibrous type I collagen is probably due to the speculative production of the free radicals in our experimental conditions. To test this hypothesis, fibrous type I collagen was incubated in PBS with AAPH (2,2'azo-bis 2-aminodinopropane) a free radicals generator. AAPH induced a dramatic and dose dependent solubilization of fibrous type I collagen.