Expression and immunohistochemical localization of leptin receptor in human periapical granuloma.

AIM: To investigate the expression and immunohistochemical localization of leptin receptor (LEPR) in human periapical granulomas. METHODOLOGY: Periapical inflammatory lesions were obtained from extracted human teeth and teeth which underwent periapical surgery. After their histopathological categorization as periapical granulomas (n = 20), they were examined by immunohistochemistry using human LEPR monoclonal antibodies. LEPR mRNA expression was also determined by quantitative real-time PCR (qRT-PCR), and the amount of LEPR protein was analysed by immunoblot. RESULTS: All granuloma samples expressed LEPR. Amongst inflammatory cells, only macrophages showed expression of LEPR. Western blot analysis revealed the presence in the samples of a protein with apparent molecular weight of ~120 kDa, corresponding to the estimated molecular weight of LEPR. The qRT-PCR analysis demonstrated the expression of LEPR mRNA, corresponding the size of the amplified fragment (338 bp), assessed by agarose gel electrophoresis, to that of LEPR mRNA. CONCLUSIONS: Human periapical granulomas express LEPR. In periapical granulomas, only macrophages showed expression of LEPR. This finding suggests that leptin can play a role in inflammatory and immune periapical responses.

¿Son aplicables los criterios analíticos generales para definir el hipotiroidismo en personas con síndrome de Down? / Are suitable general clinic criteria for defining hypothyroidism in people with Down syndrome?

Las diversas series publicadas sobre la prevalencia de las alteraciones tiroideas en las personas con síndrome de Down (SD) muestran una gran dispersión de resultados, aunque todas coinciden en señalar una frecuencia mayor que en la población general. La causa de estas diferencias puede que dependa del método de selección de la muestra. En este trabajo se estudia una población sana de adolescentes con SD, perteneciente a la Asociación de Málaga, seleccionada aleatoriamente, al margen del circuito asistencial. Al valorar la tirotropina (TSH) como parámetro bioquímico para definir la función tiroidea, resulta que la media de la distribución de la población con SD estudiada se sitúa 2 desviaciones estándar por encima de la población general. Estos datos muestran que son dos poblaciones diferentes, por lo que sería necesario definir los criterios de normalidad e hipotiroidismo subclínico, dudoso o patológico, y proponer nuevas pautas para iniciar el tratamiento (AU)

Studies on the prevalence of thyroid disorders in people with Down syndrome (DS) show a wide dispersion of results. However, most of these studies agree in indicating a greater frequency than in the general population. The cause of these differences may depend on the method of sample selection. In this work we studied a healthy population of adolescents with DS of the Association of Málaga, selected randomly and regardless of the medical care. Mean TSH distribution, used here as a tool to define the biochemical thyroid function of the studied DS population, was two standard deviation higher than the mean for the general population. These data show that in terms of TSH the DS population is a distinct population with respect to the general population. This clearly indicates that it would be necessary to identify and define new criteria to establish what is normal, subclinical hypothyroidism, borderline or pathological, and to propose new treatment guide (AU)

Aminooxy analog of histamine is an efficient inhibitor of mammalian L-histidine decarboxylase: combined in silico and experimental evidence.

Histamine plays highlighted roles in the development of many common, emergent and rare diseases. In mammals, histamine is formed by decarboxylation of L-histidine, which is catalyzed by pyridoxal-5'-phosphate (PLP) dependent histidine decarboxylase (HDC, EC 4.1.1.22). The limited availability and stability of the protein have delayed the characterization of its structure-function relationships. Our previous knowledge on mammalian HDC, derived from both in silico and experimental approaches, indicates that an effective competitive inhibitor should be capable to form an "external aldimine-like structure" and have an imidazole group, or its proper mimetic, which provides additional affinity of PLP-inhibitor adduct to the HDC active center. This is confirmed using HEK-293 cells transfected to express human HDC and the aminooxy analog of histidine, 4(5)-aminooxymethylimidazole (O-IMHA, IC50 ≈ 2 × 10(-7) M) capable to form a PLP-inhibitor complex (oxime) in the enzyme active center. Taking advantage of the availability of the human HDC X-ray structure, we have also determined the potential interactions that could stabilize this oxime in the active site of mammalian HDC.

Pharmacological potential of biogenic amine-polyamine interactions beyond neurotransmission.

Histamine, serotonin and dopamine are biogenic amines involved in intercellular communication with multiple effects on human pathophysiology. They are products of two highly homologous enzymes, histidine decarboxylase and l-aromatic amino acid decarboxylase, and transmit their signals through different receptors and signal transduction mechanisms. Polyamines derived from ornithine (putrescine, spermidine and spermine) are mainly involved in intracellular effects related to cell proliferation and death mechanisms. This review summarizes structural and functional evidence for interactions between components of all these amine metabolic and signalling networks (decarboxylases, transporters, oxidases, receptors etc.) at cellular and tissue levels, distinct from nervous and neuroendocrine systems, where the crosstalk among these amine-related components can also have important pathophysiological consequences. The discussion highlights aspects that could help to predict and discuss the effects of intervention strategies.

Leptin expression in healthy and inflamed human dental pulp.

AIM: To investigate the expression of leptin in healthy and inflamed human dental pulp. METHODOLOGY: Twenty-one pulp samples were obtained from freshly caries- and restoration-free extracted human third molars. In seven-third molars (inflamed pulp group), inflammation was induced prior to extraction. Pulp samples were processed, and leptin expression was determined by quantitative real-time PCR (qRT-PCR) and the amount of leptin by immunoblot. RESULTS: All healthy and inflamed dental pulp samples expressed leptin. Western blot analysis revealed the presence of a protein with an apparent molecular weight of ~16 kDa in human dental pulp, which corresponds to the estimated molecular weight of leptin. The expression of leptin mRNA in dental pulp was confirmed by qRT-PCR analysis, and the size of the amplified fragments (296 bp for leptin and 194 bp for cyclophilin) was confirmed by agarose gel electrophoresis. The expression of leptin in the inflamed pulp group was significantly greater (P < 0.05) than in healthy teeth. The relative amount of leptin in inflamed pulps was almost twice than in healthy pulps. CONCLUSIONS: For the first time, the presence of leptin in human dental pulp tissues has been demonstrated. The upregulation of leptin expression in inflamed pulp samples suggests that leptin can play a role in pulpal inflammatory and immune responses.

Regulatory cross-talk of mouse liver polyamine and methionine metabolic pathways: a systemic approach to its physiopathological consequences.

Both polyamines and methionine derivatives are nitrogen compounds directly related to the regulation of gene expression. In silico predictions and experimental evidence suggest a cross-talk between polyamine and methionine metabolism in mammalian tissues. Since liver is the major organ that controls nitrogen metabolism of the whole organism, it is the best tissue to further test this hypothesis in vivo. In this work, we studied the effects of the chronic administration of a methionine-supplemented diet (0.5% Met in drinking water for 5 months) on the liver of mice (designated as MET-mice). Metabolic and proteomic approaches were performed and the data obtained were subjected to biocomputational analysis. Results showed that a supplemental methionine intake can indeed regulate biogenic amine metabolism in an in vivo model by multiple mechanisms including metabolic regulation and specific gene demethylation. Furthermore, putative systemic effects were investigated by molecular and cellular biology methods. Among other results, altered expression levels of multiple inflammation and cell proliferation/death balance markers were found and macrophage activation was observed. Overall, the results presented here will be of interest across a variety of biomedical disciplines, including nutrition, orphan diseases, immunology and oncology.

Sam68 mediates leptin-stimulated growth by modulating leptin receptor signaling in human trophoblastic JEG-3 cells.

BACKGROUND: Sam68, a member of the signal transduction and activation of RNA metabolism (STAR) family of RNA-binding proteins, has been previously implicated as an adaptor molecule in different signaling systems, including leptin receptor (LEPR) signaling. LEPR activation is known to stimulate JAK-STAT, MAPK and PI3K signaling pathways, thus mediating the biological effects of leptin in different cell types, including trophoblastic cells. We have recently found that leptin stimulation also promotes the overexpression and tyrosine phosphorylation of Sam68 in human trophoblastic JEG-3 cells, suggesting a role for Sam68 in leptin signaling and action in these cells. In the present work, we have studied the participation of Sam68 in the main signaling pathways activated by LEPR to increase growth and proliferation in trophoblastic JEG-3 cells. METHODS: We used an antisense strategy to down-regulate Sam68 expression in these cells, and we studied LEPR signaling by immunoprecipitation and poly-U affinity precipitation and by analyzing phosphorylation levels of signaling proteins by immunoblot. The effect of leptin on protein synthesis and proliferation was studied by ³[H]-leucine and ³[H]-thymidine incorporation. RESULTS: Sam68 knockdown impaired leptin activation of JAK-STAT, PI3K and MAPK signaling pathways in JEG-3 cells. We have also found that leptin-stimulated Sam68 tyrosine phosphorylation is dependent on JAK-2 activity, since the pharmacological inhibitor AG490 prevents the phosphorylation of Sam68 in JEG-3 cells. Finally, the trophic and proliferative effect of leptin in trophoblastic cells is dependent on Sam68 expression, since its down-regulation impaired the leptin-stimulated DNA and protein synthesis. CONCLUSIONS: These data demonstrate that Sam68 participates in the main signaling pathways of LEPR to mediate the trophic and proliferative effect of leptin in human trophoblastic cells.

A re-evaluation of the mitogenic effect of serotonin on vascular endothelial cells.

Serotonin is an extracellular mediator recognized by seven different types of receptors, thus giving rise to pleiotropic intracellular responses. One of these responses is the activation of proliferation for a number of cell types. The induction of proliferation of otherwise quiescent endothelial cells is a key step of angiogenesis. Previously published work concerning the effect of serotonin on endothelial cell proliferation is controversial. The present work is aimed to re-evaluate the mitogenic role of serotonin on endothelial cells, since a pro-angiogenic role for serotonin could be hypothesized if its mitogenic potential on these cells were confirmed. By using three different types of endothelial cells and three experimental approaches, we demonstrate that serotonin cannot be considered a general mitogen for endothelial cells.

Molecular characterization of five patients with homocystinuria due to severe methylenetetrahydrofolate reductase deficiency.

Methylenetetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism. Disturbed function of the enzyme results in hyperhomocysteinemia and causes severe vascular and neurological disorders and developmental delay. Five patients suspected of having non-classical homocystinuria due to MTHFR deficiency were examined with respect to their symptoms, MTHFR enzyme activity and genotypes of the MTHFR gene. All patients presented symptoms of severe central nervous system disease. Two patients died, at the ages of 15 months and 14 years. One patient is currently 32 years old, and is being treated with betaine and folinic acid. The other two patients, with an early diagnosis and a severe course of the disease, are currently improving under treatment. MTHFR enzyme activity in the fibroblasts of four of the patients was practically undetectable. We found four novel mutations, three of which were missense changes c.664G> T (p.V218L), c.1316T> C (p.F435S) and c.1733T> G (p.V574G), and the fourth was the 1-bp deletion c.1780delC (p.L590CfsX72). We also found the previously reported nonsense mutation c.1420G> T (p.E470X). All the patients were homozygous. Molecular modelling of the double mutant allele (p.V218L; p.A222V) revealed that affinity for FAD was not affected in this mutant. For the p.E470X mutation, the evidence pointed to nonsense-mediated mRNA decay. In general, genotype-phenotype analysis predicts milder outcomes for patients with missense changes than for those in which mutations led to severe alterations of the MTHFR protein.

Substrate uptake and protein stability relationship in mammalian histidine decarboxylase.

There is some evidence linking the substrate entrance in the active site of mammalian histidine decarboxylase and an increased stability against proteolytic degradation. In this work, we study the basis of this relationship by means of protein structure network analysis and molecular dynamics simulations. We find that the substrate binding to the active site influences the conformation of a flexible region sensible to proteolytic degradation and observe how formation of the Michaelis-Menten complex increases stability in the conformation of this region.

Exploring polyamine regulation by nascent histamine in a human-transfected cell model.

There are multiple lines of evidence suggesting interplay between histamine and polyamines in several mammalian cell types. However, the complex metabolic context makes it difficult to elucidate the mechanisms involved. Histamine's effects can be elicited after its binding to any of the four subtypes of G-protein coupled histamine membrane receptors. In addition, intracellular histamine can also interfere with polyamine metabolism, since there are several metabolic connections between the synthesis and degradation pathways of both types of amines. In order to dissect the metabolic effects of intracellular histamine on polyamine metabolism, we chose a well-known cell culture line, i.e., the human embryonic kidney 293 cells (HEK-293 cells). Initially, we show that HEK-293 cells lack a polyamine metabolic response to extracellular histamine, even over a wide range of histamine concentrations. HEK-293 cells were transfected with active and inactive versions of human histidine decarboxylase, and changes in many of the overlapping metabolic factors and limiting steps were tested. Overall, the results indicate a regulatory effect of histamine on the post-transcriptional expression of ornithine decarboxylase and suggest that this effect is primarily responsible for the decrease in polyamine synthesis and partial blockade of cell-cycle progression, which should affect cell proliferation rate.

Structural features of mammalian histidine decarboxylase reveal the basis for specific inhibition.

For a long time the structural and molecular features of mammalian histidine decarboxylase (EC 4.1.1.22), the enzyme that produces histamine, have evaded characterization. We overcome the experimental problems for the study of this enzyme by using a computer-based modelling and simulation approach, and have now the conditions to use histidine decarboxylase as a target in histamine pharmacology. In this review, we present the recent (last 5 years) advances in the structure-function relationship of histidine decarboxylase and the strategy for the discovery of new drugs.

Study by optical spectroscopy and molecular dynamics of the interaction of acridine-spermine conjugate with DNA.

We report a spectroscopic and theoretical study of the interaction between double-stranded oligonucleotides containing either adenine-thymine or guanine-cytosine alternating sequences and N1-(Acridin-9-ylcarbonyl)-1,5,9,14,18-pentazaoctadecane, or ASC, which is formed by the covalent bonding of spermine and 9-amidoacridine moieties via a trimethylene chain. Solutions containing the oligonucleotides and the conjugate at different molar ratios were studied using complementary spectroscopic techniques, including electronic absorption, fluorescence emission, circular dichroism, and Raman spectroscopy. The spectroscopical properties of ASC at both the vibrational and the electronic levels were described by means of ab initio quantum-chemical calculations on 9-amidoacridine, used as a model compound. Molecular dynamics calculations, based on the QM/MM methodology, were also performed using previously docked structures of two oligonucleotide-ASC complexes containing the A-T and the G-C sequence. Our data, taken all together, allowed us to demonstrate that conjugation of spermine to acridine modulates and gives additional properties to the interaction of the latter with DNA. As the ASC molecule has a high affinity by the polyamine transport system, these results are promising for their application in the development of new anti-tumour drugs.

In silico analysis of arginine catabolism as a source of nitric oxide or polyamines in endothelial cells.

We use a modeling and simulation approach to carry out an in silico analysis of the metabolic pathways involving arginine as a precursor of nitric oxide or polyamines in aorta endothelial cells. Our model predicts conditions of physiological steady state, as well as the response of the system to changes in the control parameter, external arginine concentration. Metabolic flux control analysis allowed us to predict the values of flux control coefficients for all the transporters and enzymes included in the model. This analysis fulfills the flux control coefficient summation theorem and shows that both the low affinity transporter and arginase share the control of the fluxes through these metabolic pathways.

(-)-Epigallocatechin-3-gallate interferes with mast cell adhesiveness, migration and its potential to recruit monocytes.

Mast cells are multipotent effector cells of the immune system. They are able to induce and enhance angiogenesis via multiple pathways. (-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea and a putative chemopreventive agent, was reported to inhibit tumor invasion and angiogenesis, processes that are essential for tumor growth and metastasis. Using the human mast cell line HMC-1 and commercial cDNA macroarrays, we evaluated the effect of EGCG on the expression of angiogenesis-related genes. Our data show that among other effects, EGCG treatment reduces expression of two integrins (alpha5 and beta3) and a chemokine (MCP1), resulting in a lower adhesion of mast cells associated with a decreased potential to produce signals eliciting monocyte recruitment. These effects on gene expression levels are functionally validated by showing inhibitory effects in adhesion, aggregation, migration and recruitment assays.

Development of an expression macroarray for amine metabolism-related genes.

Cationic amino acids are the precursors of biogenic amines, histamine from histidine, and putrescine, spermidine and spermine from arginine/ornithine (and methionine), as well as nitric oxide. These amines play important biological roles in inter- and intracellular signaling mechanisms related to inflammation, cell proliferation and neurotransmission. Biochemical and epidemiological relationships between arginine-derived products and histamine have been reported to play important roles in physiopathological problems. In this communication, we describe the construction of an expression macroarray containing more than 30 human probes for most of the key proteins involved in biogenic amines metabolisms, as well as other inflammation- and proliferation-related probes. The array has been validated on human mast HMC-1 cells. On this model, we have got further support for an inverse correlation between polyamine and histamine synthesis previously observed on murine basophilic models. These tools should also be helpful to understand the amine roles in many other inflammatory and neoplastic pathologies.

Polyamines: metabolism to systems biology and beyond.

Polyamines and the metabolic and physiopathological processes in which they are involved represent an active field of research that has been continuously growing since the seventies. In the last years, the trends in the focused areas of interest within this field since the 1970s have been confirmed. The impact of "-omics" in polyamine research remains too low in comparison with its deep impact on other biological research areas. These high-throughput approaches, along with systems biology and, in general, more systemic and holistic approaches should contribute to a renewal of this research area in the near future.

The usefulness of post-genomics tools for characterization of the amine cross-talk in mammalian cells.

Evidence is growing in favour of a relationship between cancer and chronic inflammation, and particularly of the role of a polyamine and histamine metabolic interplay involved in these physiopathological problems, which are indeed highly complex biological systems. Decodification of the complex inter- and intra-cellular signalling mechanisms that control these effects is not an easy task, which must be helped by systems biology technologies, including new tools for location and integration of database-stored information and predictive mathematical models, as well as functional genomics and other experimental molecular approaches necessary for hypothesis validation. We review the state of the art and present our latest efforts in this area, focused on the amine metabolism field.