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Identification and characterization of new plasmatic isoforms of the tumor suppressor gene PTPRG and development of new specific monoclonal antibodies
Full text linkObiettivi: 1) identificare la presenza e caratterizzare le diverse isoforme della proteina plasmatica di tipo recettore tirosin fosfatasi Gamma (PTPRG) 2) caratterizzazione di nuovi anticorpi monoclonali specifici per il dominio extracellulare della proteina.
Background: PTPRG è un enzima ampiamente espresso, si ritrova principalmente espresso nel polmone, stomaco, esofago, colon, fegato, milza e reni. PTPRG mappa nel CHR 3p14.2/21 ed è stato implicato come candidato gene oncosoppressore. Sono state riportate perdita di eterozigosità, delezione genica, mutazione puntiforme e ipermetilazione del promotore. La maggior parte dei recettori intracellulari possiedono domini fosfatasici simili, mentre la porzione extracellulare delle fosfatasi mostra massima variazione strutturale.
Per quel che riguarda PTPRG, nel cervello di ratto sono state riportate diverse isoforme, tra cui la sola regione extracellulare della proteina facendoci presupporre la possibilità che potesse esistere anche nell’uomo un'isoforma solubile. Una prima evidenza sperimentale dell’esistenza di questa possibile isoforma di PTPRG è già stata messa in luce nel nostro gruppo, in studi precedenti ,dove è stata evidenziata all'interno della colloide della tiroide. Tali presupposti ci hanno suggerito la possibilità che potesse esistere una forma solubile extracellulare di PTPRG anche all’interno di altri fluidi biologici.
Metodi: immunoprecipitazione, western blotting, citometria a flusso.
Risultati: Abbiamo identificato, nel plasma umano e murino, diverse nuove isoforme di PTPRG, una delle quali caratterizzata in precedenza tramite clonazione solo nel ratto. I polipeptidi identificati hanno un peso molecolare apparente di circa 120 kDa (banda principale) e di 90 kDa (banda minore) rispettivamente. Sorprendentemente, anche la proteina di lunghezza intera, che include la regione transmembrana, è stata identificata nel siero, anche se dopo ultracentrifugazione a 100000 × g, risulta precipitare in forma di soluto suggerendo la presenza di un modulo associato a vescicole di tipo esosomiale. La banda maggiore (120 kDa) è stata individuata nel fegato di topo (isolato dopo perfusione in soluzione salina in modo da eliminare eventuali tracce di plasma) e in terreno condizionato senza siero derivato da HepG2, una linea cellulare di carcinoma hepato-cellulare comunemente utilizzata per lo studio della regolamentazione della sintesi proteica epatica. L’ isoforma da 120 kDa risulta essere up-regolata dopo trattamento con etanolo e potassio dicromato, due composti noti per essere citotossici per il fegato. Con l'obiettivo futuro di creare un saggio ELISA per lo screening rapido dell’ espressione di PTPRG solubile in varie condizioni fisiopatologiche, abbiamo sviluppato e caratterizzato in seguito, due nuovi anticorpi monoclonali che riconoscono un’epitopo nel dominio extracellulare del PTPRG.
Conclusione: Abbiamo descritto per la prima volta nell'uomo e nel topo la presenza diverse isoforme solubili di PTPRG all’interno del plasma che sembrano essere rilasciate principalmente dal fegato. La banda più importante di kDa 120 sembra essere glicosilata e la sua espressione è modulata da stimoli noti per la produzione di danno epatico.
La produzione e la caratterizzazione di nuovi anticorpi monoclonali in grado di riconoscere specificamente questa proteina qui descritta consentiranno la messa a punto di nuovi test immunologici e apriranno la strada ad una migliore caratterizzazione di questa proteina e del suo ruolo nella salute e nella malattia.Aims: 1) Identify the presence and characterize the plasmatic isoforms of Receptor Type Protein Tyrosine Phosphatase Gamma (PTPRG) 2) Characterization of new monoclonal antibodies specific for the extracellular domain of the protein.
Background: PTPRG is a broadly expressed enzyme mainly expressed in lung, stomach, esophagus, colon, liver, spleen and kidney. PTPRG maps to chr 3p14.2/21 and has been implicated as a candidate tumor suppressor gene. Loss of eterozigosity, gene deletion, point mutation and promoter hypermethylation has been reported. The majority of the receptor PTP share similar intracellular domains while the extracellular portion exhibits broad structural variation. Rat brain has been reported to express various PTPRG isoforms, including the isolated extracellular region 1. Previous work from our group suggested the production on an extracellular soluble form of PTPRG in thyroid.
Methods: immunoprecipitation, western blotting, flow cytometry.
Results: We have identified, in human and murine plasma, new PTPRG isoforms, one of which previously characterized by cDNA cloning only in rat . The polypeptides identified have an apparent MW of about 120 kDa (major band) and 90 kDa (minor band) respectively. Surprisingly, also the full length protein, that include a transmembrane region, was identified in the serum and was present in the fraction precipitating at 100000 ×g, suggesting the presence of an exosome-associated form. The major band (120 kDa) was also identified in mouse liver (isolated after saline perfusion in order to remove plasma) and in serum-free conditioned medium derived from HepG2, an hepatocellular carcinoma cell line commonly used for the study of the regulation of hepatic protein synthesis. The 120 kDa isoform was up-regulated by treatment with ethanol and potassium dichromate, two compounds known to be cytotoxic for liver. With the future goal to set up an ELISA-based assay for the rapid screening of soluble PTPRG expression in various physiopathological conditions, we have developed and characterized two new monoclonal antibodies raised against the extracellular domain of PTPRG expressed in eukariotic cells (HEK 293F).
Conclusion: We have described for the first time in human and mouse the presence of soluble, plasmatic forms of PTPRG that appear to be released primarily by liver. The major 120 kDa band appear to be glycosylated and its expression is modulated by stimuli known to produce liver injury.
The production and initial characterization of new monoclonal antibodies capable to specifically recognize this protein here described will permit the set up of new immunoassays and will pave the way to a better characterization of this proteins and its role in health and disease
Identification of Protein Tyrosine Phosphatase Receptor Gamma Extracellular Domain (sPTPRG) as a Natural Soluble Protein in Plasma
Full text linkPTPRG is a widely expressed protein tyrosine phosphatase present in various isoforms. Peptides from its extracellular domain have been detected in plasma by proteomic techniques. We aim at characterizing the plasmatic PTPRG (sPTPRG) form and to identify its source.The expression of sPTPRG was evaluated in human plasma and murine plasma and tissues by immunoprecipitation and Western blotting. The polypeptides identified have an apparent Mr of about 120 kDa (major band) and 90 kDa (minor band) respectively. Full length PTPRG was identified in the 100.000×g pelleted plasma fraction, suggesting that it was present associated to cell-derived vesicles (exosomes). The release of sPTPRG by HepG2 human hepatocellular carcinoma cell line was induced by ethanol and sensitive to metalloproteinase and not to Furin inhibitors. Finally, increased levels of the plasmatic ∼120 kDa isoform were associated with the occurrence of liver damage.These results demonstrate that sPTPRG represent a novel candidate protein biomarker in plasma whose increased expression is associated to hepatocyte damage. This observation could open a new avenue of investigation in this challenging field
Characterization of new monoclonal antibodies specific for the extracellular domain of PTPRG, a candidate tumour suppressor gene
No full textNo abstract availabl
High-dilution effects revisited. 1. Physicochemical aspects
No full textSeveral lines of evidence suggest that homeopathic high dilutions (HDs) can effectively have a pharmacological action, and so cannot be considered merely placebos. However, until now there has been no unified explanation for these observations within the dominant paradigm of the dose-response effect. Here the possible scenarios for the physicochemical nature of HDs are reviewed. A number of theoretical and experimental approaches, including quantum physics, conductometric and spectroscopic measurements, thermoluminescence, and model simulations investigated the peculiar features of diluted/succussed solutions. The heterogeneous composition of water could be affected by interactive phenomena such as coherence, epitaxy and formation of colloidal nanobubbles containing gaseous inclusions of oxygen, nitrogen, carbon dioxide, silica and, possibly, the original material of the remedy. It is likely that the molecules of active substance act as nucleation centres, amplifying the formation of supramolecular structures and imparting order to the solvent. Three major models for how this happens are currently being investigated: the water clusters or clathrates, the coherent domains postulated by quantum electrodynamics, and the formation of nanoparticles from the original solute plus solvent components. Other theoretical approaches based on quantum entanglement and on fractal-type self-organization of water clusters are more speculative and hypothetical. The problem of the physicochemical nature of HDs is still far from to be clarified but current evidence strongly supports the notion that the structuring of water and its solutes at the nanoscale can play a key role
A dynamic network model of the similia principle
No full textThe use of drugs in high dilutions and the principle of similarity (or "similia") are two basic tenets of homeopathy. However, the plausibility of both is a subject of debate. Although several models have been proposed to explain the similia principle, it can be best understood and appreciated in the framework of complexity science and dynamic systems theory. This work applies a five-node Boolean network to show how self-organization and adaptation are relevant to rationalizing this traditional medical principle. Simulating the trajectories and attractors of the network system in the energy state-space provides a rudimentary and qualitative illustration of how targeted external perturbations can have pathological effects, leading to permanent, self-sustaining alterations. Similarly, changes that conversely enable the system to find its way back to the original state can induce therapeutic effects, by causing specific shifts in attractors when suitable conditions are satisfied. Extrapolating these mechanisms to homeopathy, we can envisage how major changes in the evolution of homeodynamic systems (and, eventually, healing of the entire body) can be achieved through carefully selected remedies that reproduce the whole symptom pattern of the ill state
Effects of Gelsemium sempervirens L. on pathway-focused gene expression profiling in neuronal cells
No full textETHNOPHARMACOLOGICAL RELEVANCE:
Gelsemium sempervirens L. (G. sempervirens) is a traditional medicinal plant mainly distributed in the southeastern of the United States, employed in phytotheraphy and homeopathy as nervous system relaxant to treat various types of anxiety, pain, headache and other ailments. Although animal models showed its effectiveness, the mechanisms by which it might operate on the nervous system are largely unknown.
AIM OF THE STUDY:
This study investigated for the first time by a real-time PCR technique (RT-PCR Array) the gene expression of a panel of human neurotransmitter receptors and regulators, involved in neuronal excitatory signaling, on a neurocyte cell line.
MATERIALS AND METHODS:
Human SH-SY5Y neuroblastoma cells were exposed for 24h to G. sempervirens at 2c and 9c dilutions (i.e. 2 and 9-fold centesimal dilutions from mother tincture) and the gene expression profile compared to that of cells treated with control vehicle solutions.
RESULTS:
Exposure to the G. sempervirens 2c dilution, containing a nanomolar concentration of active principle gelsemine, induced a down-regulation of most genes of this array. In particular, the treated cells showed a statistically significant decrease of the prokineticin receptor 2, whose ligand is a neuropeptide involved in nociception, anxiety and depression-like behavior.
CONCLUSIONS:
Overall, the results indicate a negative modulation trend in neuronal excitatory signaling, which can suggest new working hypotheses on the anxiolytic and analgesic action of this plant
Effects of Gelsemium sempervirens L. on pathway-focused gene expression profiling in neuronal cells
Full text linkGelsemium sempervirens L. is a traditional medicinal plant mainly distributed in the southeastern of the United States, employed in phytotheraphy and homeopathy as nervous system relaxant to treat various types of anxiety, pain, headache and other ailments. Although animal models showed its effectiveness, the mechanisms by which it might operate on the nervous system are largely unknown. This study investigated for the first time by a real-time PCR technique (RT-PCR Array) the gene expression of a panel of human neurotransmitter receptors and regulators, involved in neuronal excitatory signaling, on a neurocyte cell line. Materials and methods: Human SH-SY5Y neuroblastoma cells were exposed for 24 h to Gelsemium sempervirens at 2c and 9c dilutions (i.e. 2 and 9-fold centesimal dilutions from mother tincture) and the gene expression profile compared to that of cells treated with control vehicle solutions. Results: Exposure to the Gelsemium sempervirens 2c dilution, containing a nanomolar concentration of active principle gelsemine, induced a down-regulation of most genes of this array. In particular, the treated cells showed a statistically significant decrease of the prokineticin receptor 2, whose ligand is a neuropeptide involved in nociception, anxiety and depression-like behavior. Conclusions: Overall, the results indicate a negative modulation trend in neuronal excitatory signaling, which can suggest new working hypotheses on the anxiolytic and analgesic action of this plant
Distribution of different isoforms of receptor protein tyrosine phosphatase γ (Ptprg-RPTP γ) in adult mouse brain: upregulation during neuroinflammation.
Full text linkThe receptor protein tyrosine phosphatase γ (Ptprg-RPTPγ) is a receptor protein widely expressed in many tissues, including the central nervous system (CNS). Several RPTPγ isoforms are expressed in the brain during development and in adulthood, but their distribution and role are unknown. In this study, we investigated the distribution of some RPTPγ isoforms in the adult brain using antibodies against the epitopes localized in the C- and in the N-terminal domains of the full length isoform of RPTPγ. We found a predominant and widespread neuronal positivity throughout the neocortex, hippocampus, striatum and in many nuclei of the brainstem and cerebellum. At least 2 distinct isoforms that can co-exist in various compartments in the same cell are detectable in different neuron types. Immunopositivity for epitopes located in both the N- and C-terminus domains were found in the neuropil of cortical and hippocampal neurons, whereas the N-terminal domain positivity was found in the soma, often without colocalization with its C-terminal counterpart. Among glial cells, some protoplasmic and perivascular astrocytes and the cerebellar Bergmann glia, express RPTPγ. The astrocytic expression of RPTPγ and putative processing isoforms of 120 and 80 kDa increases during neuroinflammation, in particular 24 h after LPS treatment. Activated astrocytes were found to be strongly positive for RPTPγ also in a mice model of Alzheimer's disease. Our results confirm previous findings and enrich the current knowledge of RPTPγ distribution in the CNS, highlighting a role of RPTPγ during neuroinflammation processes
High-dilution effects revisited. 2. Pharmacodynamic mechanisms
No full textThe pharmacodynamics aspects of homeopathic remedies are appraised by laboratory studies on the biological effects at various levels (cellular,molecular and systemic). The major question is how these medicines may work in the body. The possible answers concern the identification of biological targets,the means of drug-receptor interactions,the mechanisms of signal transmission and amplification,and the models of inversion of effects according to the traditional 'simile' rule. These problems are handled by two experimental and theoretical lines,according to the doses or dilutions considered (low-medium versus high dilutions). Homeopathic formulations in low-medium dilutions,containing molecules in the range of ultra-low doses,exploit the extreme sensitivity of biological systems to exogenous and endogenous signals. Their effects are interpreted in the framework of hormesis theories and paradoxical pharmacology. The hypotheses regarding the action mechanisms of highly diluted/dynamized solutions (beyond Avogadro-Loschmidt limit) variously invoke sensitivity to bioelectromagnetic information,participation of water chains in signalling,and regulation of bifurcation points of systemic networks. High-dilution pharmacology is emerging as a pioneering subject in the domain of nanomedicine and is providing greater plausibility to the puzzling claims of homeopathy
Cell sensitivity, non-linearity and inverse effects
No full textIt has been claimed that the homeopathic principle of ‘similarity’ (or ‘similia’) and the use of individualized remedies in extremely low doses conflicts with scientific laws, but this opinion can be disputed on the basis of recent scientific advances. Several mechanisms to explain the responsiveness of cells to ultra-low doses and the similarity as inversion of drug effects, have again been suggested in the framework of hormesis and modern paradoxical pharmacology. Low doses or high dilutions of a drug interact only with the enhanced sensitivities of regulatory systems, functioning as minute harmful stimuli to trigger specific compensatory healing reactions. Here we review hypotheses about homeopathic drug action at cellular and molecular levels, and present a new conceptual model of the principle of similarity based on allosteric drug action. While many common drugs act through orthostatic chemical interactions aimed at blocking undesired activities of enzymes or receptors, allosteric interactions are associated with dynamic conformational changes and functional transitions in target proteins, which enhance or inhibit specific cellular actions in normal or disease states. The concept of allostery and the way it controls physiological activities can be broadened to include diluted/dynamized compounds, and may constitute a working hypothesis for the study of molecular mechanisms underlying the inversion of drug effects
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