Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin.

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB1.

Diagnostic efficacy of model-based iterative reconstruction algorithm in an assessment of coronary artery in comparison with standard hybrid-Iterative reconstruction algorithm: dose reduction and image quality.

PURPOSE: To evaluate the image quality and radiation dose exposure of low-dose coronary CTA (cCTA) study, reconstructed with the new model-based iterative reconstruction algorithm (IMR), compared with standard hybrid-iterative reconstruction (iDose4) cCTA in patients with suspected coronary artery disease. MATERIALS AND METHODS: Ninety-eight patients with an indication for coronary CT study were prospectively enrolled. Fifty-two patients (study group) underwent 256-MDCT low-dose cCTA (80 kV; automated-mAs; 60 mL of CM, 350 mgL/mL) with prospective ECG-triggering acquisition and IMR. A control group of 46 patients underwent 256-MDCT standard prospective ECG-gated protocol (100 kV; automated-mAs; 70 mL of CM, 400 mgL/mL; iDose4). Subjective and objective image quality (attenuation value, SD, SNR and CNR) were evaluated by two radiologists subjectively. Radiation dose exposure was quantified as DLP, CTDIvol and ED. RESULTS: Mean values of mAs were significantly lower for IMR-cCTA (167 ± 62 mAs) compared to iDose-cCTA (278 ± 55 mAs), p < 0.001. With a significant reduction of 38% in radiation dose exposure (DLP: IMR-cCTA 91.7 ± 26 mGy cm vs. iDose-cCTA 148.6 ± 35 mGy cm; p value < 0.001), despite the use of different CM, we found higher mean attenuation values of the coronary arteries in IMR group compared to iDose4 (mean density in LAD: 491HU IMR-cCTA vs. 443HU iDose-cCTA; p = 0.03). We observed a significant higher value of SNR and CNR in study group due to a lower noise level. Qualitative analysis did not reveal any significant differences between the two groups (p = 0.23). CONCLUSIONS: Low-dose cCTA study combined with IMR reconstruction allows to correctly evaluate coronary arteries disease, offering high-quality images and significant radiation dose exposure reduction (38%), as compared to standard cCTA protocol.

Granulomatosis with polyangiitis presenting with diffuse alveolar hemorrhage requiring extracorporeal membrane oxygenation with rapid multiorgan relapse: A case report.

RATIONALE: Granulomatosis with polyangiitis (GPA) is an antineutrophil cytoplasmatic antibodies (ANCA)-associated vasculitis affecting small- and medium-sized blood vessels, mostly involving lung and kidney. PATIENT CONCERNS: We report the case of a 33-year-old man that presented with acute respiratory distress syndrome caused by alveolar hemorrhage. DIAGNOSES: Aggressive GPA presenting with diffuse alveolar hemorrhage and multiorgan involvement. INTEVENTIONS: Immunosuppressive therapy, plasma exchange, extracorporeal membrane oxygenation (ECMO). OUTCOMES: Relapse occurred very early, despite immunosuppressive treatment, with a rare involvement of genital system (epididymitis) and rapidly progressive glomerulonephritis difficult to treat. LESSONS: GPA is a challenging, multifaceted disease that can require aggressive supportive therapy and is associated with a high rate of relapse that may present with uncommon site of involvement.

Quercetin: A flavonol with multifaceted therapeutic applications?

Great interest is currently centered on the biologic activities of quercetin a polyphenol belonging to the class of flavonoids, natural products well known for their beneficial effects on health, long before their biochemical characterization. In particular, quercetin is categorized as a flavonol, one of the five subclasses of flavonoid compounds. Although flavonoids occur as either glycosides (with attached glycosyl groups) or as aglycones, most altogether of the dietary intake concerning quercetin is in the glycoside form. Following chewing, digestion, and absorption sugar moieties can be released from quercetin glycosides. Several organs contribute to quercetin metabolism, including the small intestine, the kidneys, the large intestine, and the liver, giving rise to glucuronidated, methylated, and sulfated forms of quercetin; moreover, free quercetin (such as aglycone) is also found in plasma. Quercetin is now largely utilized as a nutritional supplement and as a phytochemical remedy for a variety of diseases like diabetes/obesity and circulatory dysfunction, including inflammation as well as mood disorders. Owing to its basic chemical structure themost obvious feature of quercetin is its strong antioxidant activity which potentially enables it to quench free radicals from forming resonance-stabilized phenoxyl radicals. In this review the molecular, cellular, and functional bases of therapy will be emphasized taking strictly into account data appearing in the peer-reviewed literature and summarizing the main therapeutic applications of quercetin; furthermore, the drug metabolism and the main drug interaction as well as the potential toxicity will be also spotlighted.

Perfusion computed tomography in patients with hepatocellular carcinoma treated with thalidomide: initial experience.

OBJECTIVE: The objective of the study was to evaluate the role of perfusion computed tomography (CT) for monitoring and predicting therapy response in patients with hepatocellular carcinoma treated with thalidomide. METHODS: Twenty-four patients with advanced hepatocellular carcinoma were treated with thalidomide. Perfusion and conventional CT were performed at baseline and every 2 months until disease progression. Baseline tumor size and enhancement characteristics, as well as baseline perfusion parameters and their changes after therapy, were explored and tested for association with therapy response. RESULTS: Perfusion CT was feasible in 18 patients. Baseline tumor size and enhancement characteristics showed no predictive value, whereas baseline blood flow and blood volume were higher in patients with progressive disease (P < 0.042), with cutoff values for blood flow (16.7 mL/100 g per minute) and blood volume (1.84 mL/100 g) predicting progressive disease in 83.3% and 77.8% of patients, respectively. Significant changes were observed after 2, 4, and 6 months in blood flow (P < 0.031), blood volume after 4 months (P = 0.018), and mean transit time after 4 and 6 months (P = 0.030) in patients with stable disease at 6 months. CONCLUSIONS: Baseline blood flow and blood volume predicted response to therapy in our cohort.

Pycnogenol: a blend of procyanidins with multifaceted therapeutic applications?

Great interest is currently centred on the biologic activities of pycnogenol a standardized plant extract obtained from the bark of the French maritime pine Pinus pinaster (formerly known as Pinus maritima), Aiton, subspecies Atlantica des Villar (Pycnogenol, Horphag Research Ltd., UK, Geneve, Switzerland), which grows in the coastal southwest France. The quality of this extract is specified in the United States Pharmacopeia (USP 28). Between 65% and 75% of Pycnogenol are procyanidins comprising of catechin and epicatechin subunits with varying chain lengths. Other constituents are polyphenolic monomers, phenolic or cinnamic acids and their glycosides. As many studies indicate, pycnogenol components are highly bioavailable. Uniquely, pycnogenol displays greater biologic effects as a mixture than its purified components do individually indicating that the components interact synergistically. Pycnogenol is now utilized throughout the world as a nutritional supplement and as a phytochemical remedy for various diseases ranging from chronic inflammation to circulatory dysfunction, including several impaired psycho-physiological functions. Owing to the basic chemical structure of its components, the most obvious feature of pycnogenol is its strong antioxidant activity. In fact, phenolic acids, polyphenols, and in particular flavonoids, are composed of one (or more) aromatic rings bearing one or more hydroxyl groups and are therefore potentially able to quench free radicals by forming resonance-stabilized phenoxyl radicals. In this review, emphasizing the molecular, cellular, and functional bases of therapy, data appearing in the peer-reviewed literature and focussing the main therapeutic applications of pycnogenol will be summarized and critically evaluated.

Resveratrol: a natural polyphenol with multiple chemopreventive properties.

Resveratrol, a naturally occurring polyphenol, shows pleiotropic health beneficial effects, including anti-oxidant, anti-inflammatory, anti-aging, cardioprotective and neuroprotective activities. Due to the several protective effects and since this compound is widely distributed in the plant kingdom, resveratrol can be envisaged as a chemo-preventive/curative agent introduced almost daily with the diet. Currently, a number of preclinical findings suggest resveratrol as a promising nature's weapon for cancer prevention and treatment. A remarkable progress in elucidating the molecular mechanisms underlying anti-cancer properties of resveratrol has been achieved in the last years. Concerning the resveratrol mechanism of action as a protective (vs. normal cells and tissues) and toxic (vs. cancer cells) compound, many studies focus on its antioxidant capacity as well as on its ability to trigger and favor the apoptotic cascade in malignant cells. However, a generalized mechanism of action able to explain this dual effect of resveratrol has not yet been clearly established. In addition to these important functions, resveratrol is reported to exhibit several other biological/biochemical protective effects on heart, circulation, brain and age-related diseases which are summarized in this Review.

Perfusion computed tomography for monitoring induction chemotherapy in patients with squamous cell carcinoma of the upper aerodigestive tract: correlation between changes in tumor perfusion and tumor volume.

OBJECTIVE: The aim of this study was to assess the potential of perfusion computed tomography (CTp) for monitoring induction chemotherapy in patients with squamous cell carcinoma (SCCA) of the upper aerodigestive tract. MATERIALS AND METHODS: Twenty-five patients with advanced SCCA underwent CTp and volumetric CT before and after induction chemotherapy. Perfusion CT parameters were calculated in the tumor, normal tissue, and muscles and correlated with tumor volume. RESULTS: The blood flow (BF), blood volume (BV), and permeability surface were significantly higher, and the mean transit time was significantly lower in the tumor than in the normal tissue. The tumor BF and BV significantly decreased, and the mean transit time significantly increased after the therapy; decrease in BF and BV correlated with tumor volume reduction after chemotherapy. The baseline tumor BV was significantly lower in nonresponders compared with that in responders. CONCLUSIONS: In patients with SCCA, CTp showed potential for monitoring induction chemotherapy, reduction in tumor BF and BV correlated with reduction of tumor volume after chemotherapy, and baseline tumor BV may predict response to chemotherapy.

AZT: an old drug with new perspectives.

The science of antiviral research was well advanced when HIV/AIDS appeared as a major new virus disease in the early 1980s. The first effective antiviral compound (AZT, azidothymidine, zidovudine) was already among the library of compounds screened and was promptly reported to be a specific inhibitor of retroviruses, including HIV. Due to the pivotal role of AZT in HIV treatment, this review summarizes the most known effects -some of which are toxic side effects- induced by AZT a drug which is still used in the combined therapy of HIV-infected patients. Among the toxic side effects, a severe bone marrow toxicity manifested as anemia, neutropenia and siderosis, and caused by inhibition of heme and globin synthesis together with a general derangement of iron supply, have been reported. In this regard, we proved that while AZT and its monophosphorylated derivative AZTMP were unable to chelate iron, the triphosphate form AZTTP displayed a significant capacity to remove iron from transferrin. Moreover, we have previously demonstrated that AZT-exposed K562 cells showed an increase of transferrin receptors located on the cell membrane without affecting their biosynthesis, but slowing down their endocytotic pathway. Interestingly, literature data report the impairement of glycosylation reactions by AZT. Indeed, we have shown that AZT-treated K562 cells exhibited a reduced sialylation of proteins and lipids, and a strong inhibition of alpha,(2-->8) sialyltransferase activity while beta,(1-->4)galactosyltransferase and beta-galactosidase activities were significantly increased. These latter observations could be of clinical relevance since alterations of intracellular and cell surface carbohydrate expression and composition, often are associated with several diseases. However, contrarily to previous reports by other authors on AZT as an inhibitor of plant and bacterial toxins activity, we have demonstrated that AZT not only did not inhibit saporin toxicity, but even increased the cytotoxic activity of this plant toxin on K562 cells. Furthermore, the review enlightens the potential utilization of AZT as a tool in proteomics since in the recent years several genes responding to this drug have been identified in different cell lines. We have shown, for the first time, an over-expression of two proteins (PDI-A3 and sthatmin), and a full repression of two others (HSP-60 and SOD1) in AZT-exposed K562 cells. At present, we are investigating if the above reported alterations are a general feature of AZT-treatment of cultured cells, or they represent a peculiar characteristic of a specific cell line. Finally, the paper reviews a number of novel methodologies aimed at enhancing the AZT plasma levels and its bioavailability in all human organs in order to improve its therapeutic efficacy against HIV infection. These new possibilities, namely the AZT prodrug strategy, the AZT transdermal delivery and the targeted brain delivery, are yet not in use for humans but they are under experimental studies.

First synthetic probe for the detection and quantification of a protein with a potential alpha,(2-->8)sialyltransferase activity.

There is considerable interest in monitoring alpha,(2-->8)sialyltransferase (ST8) levels; however, there are few specific and sensitive methods to directly detect and quantitate the protein. This paper reports the development of a synthetic probe composed of oxidized colominic acid coupled to biotinyl-L-lysine hydrazide to detect and quantify ST8 with putative "initiase" activity and its use in three solid-phase applications. The detection limit observed for ST8 purified from K562 cells was approximately 2 pg by dot-blot analysis. In Western blots the probe bound and specifically recognized a protein band corresponding to ST8. In ELISA a linear dose response was obtained for pure protein in the range of 50-200 pg. Analysis of 3'-azido-3'deoxythymidine-treated cells by all three methods showed a reduction in ST8 compared to control cells; treated cells had 73% of control levels by ELISA. This probe will be useful for studies on the expression ST8 and its role in glycoconjugate biosynthesis.

Effects of AZT on cellular iron homeostasis.

3'-azido-3'-deoxythymidine (AZT), the first chemotherapeutic drug approved by FDA for treatment of HIV-infected patients and still used in combination therapy, has been shown to induce, upon prolonged exposure, severe bone marrow toxicity manifested as anemia, neutropenia and siderosis. These toxic effects are caused by inhibition of heme synthesis and, as a consequence, transferrin receptor (TfR) number appears increased and so iron taken up by cells. Since iron overload can promote the frequency and severity of many infections, siderosis is viewed as a further burden for AIDS patients. We have previously demonstrated that AZT-treated K562 cells showed an increase of the number of TfRs located on the surface of the plasma membrane without affecting their biosynthesis, but slowing down their endocytotic pathway. In spite of the higher number of receptors on the plasma-membrane of AZT-treated cells, intracellular accumulation of iron showed a similar level in control and in drug-exposed cells. The chelating ability of AZT and of its phosphorylated derivatives, both in an acellular system and in K562 cells, was also checked. The results demonstrated that AZT and AZTMP were uneffective as iron chelators, while AZTTP displayed a significant capacity to remove iron from transferrin (Tf). Our results suggest that AZT may be not directly involved in the iron overloading observed upon its prolonged use in AIDS therapy. The iron accumulation found in these patients is instead caused by other unknown mechanisms that need further studies to be clarified.

Protein glycans alteration and a different distribution of some enzymatic activities involved in the glycan processing are found in AZT-treated K562 cells.

In this paper we report that 3'-azido-3'-deoxythymidine (AZT) treatment of human erythroleukemia (K562) cells greatly alters the pattern of protein glycans and significantly modifies beta,(1 --> 4)galactosyltransferase, beta-galactosidase, and alpha,(2 --> 8)sialyltransferase activities. In particular, AZT-treated K562 cells exhibited a decreased incorporation of sialic acid (86% of control) into protein glycans, being the reduced alpha,(2 --> 6) incorporation almost of the same magnitude with respect to that of alpha,(2 --> 3) (93 and 90% of control, respectively). Moreover, the drug exposure of cells induced a decrease of both mannose terminally linked and galactose linked as beta,(1 --> 4) (90 and 92% of control, respectively) and a significant increase of galactose beta,(1 --> 3) (112% of control). In addition, beta,(1 --> 4)galactosyltransferase and beta-galactosidase activities were found enhanced in K562-treated cells (30 and 12%, respectively), while alpha,(2-8 )sialyltransferase activity decreased (75% of control). Sialyltransferase activities of other types i.e. 30, 60, 3 N, 6 N, did not show any appreciable differences irrespective of AZT-treatment. Besides previous studies which report that AZT exposure of K562 cells, indirectly prevents nucleotide-sugar import into the Golgi complex, with consequent inhibition of glycosylation, our observations show for the first time that AZT affects several enzymatic activities involved in specific glycosylation reactions leading, in turn, to protein glycans alteration.

Osteopontin modulates prostate carcinoma invasive capacity through RGD-dependent upregulation of plasminogen activators.

Osteopontin, a non-collageneous bone matrix protein, is produced in several human tumors but its role in cancer progression has been only partially elucidated. In this study we investigated the potential role of osteopontin in the malignancy of prostate cancer cells. Chemotaxis and chemoinvasion analyses revealed a dose-dependent increase in PC3 cell movement induced by osteopontin and a strict dependence of cell invasion on alphavbeta3 integrin function. The pattern of protease expression was modified by osteopontin and was characterized by an upregulation of plasminogen activators. Our findings suggest that osteopontin may confer selective malignant potential to prostate cancer cells through the enhancement of their invasive and proteolytic capability.