Finding of endocannabinoids in human eye tissues: implications for glaucoma

Cannabinoid CB(1) receptors are involved in ocular physiology and may regulate intraocular pressure (IOP). However, endocannabinoid levels in human ocular tissues of cornea, iris, ciliary body, retina, and choroid from normal and glaucomatous donors have not been investigated. Anandamide (N-arachidonoylethanolamine; AEA), 2-arachidonoylglycerol (2-AG), and the anandamide congener, palmitoylethanolamide (PEA), were detected in all the human tissues examined. In eyes from patients with glaucoma, significantly decreased 2-AG and PEA levels were detected in the ciliary body, an important tissue in the regulation of IOP. The findings suggest that these endogenous compounds may have a role in this disease, particularly with respect to regulation of IOP.     

Expression pattern of receptor activator of NFκB (RANK) in a series of primary solid tumors and related bone metastases

Receptor activator of NFκB ligand (RANKL), RANK, and osteoprotegerin (OPG) represent the key regulators of bone metabolism both in normal and pathological conditions, including bone metastases. To our knowledge, no previous studies investigated and compared RANK expression in primary tumors and in bone metastases from the same patient. We retrospectively examined RANK expression by immunohistochemistry in 74 bone metastases tissues from solid tumors, mostly breast, colorectal, renal, lung, and prostate cancer. For 40 cases, tissue from the corresponding primary tumor was also analyzed. Sixty‐six (89%) of the 74 bone metastases were RANK‐positive and, among these, 40 (59.5%) showed more than 50% of positive tumor cells. The median percentage of RANK‐positive cells was 60% in primary tumors and metastases, without any statistically significant difference between the two groups (P = 0.194). The same percentage was obtained by considering only cases with availability of samples both from primary and metastasis. Our study shows that RANK is expressed by solid tumors, with high concordance between bone metastasis and corresponding primary tumor. These data highlight the central role of RANK/RANKL/OPG pathway as potential therapeutic target not only in bone metastasis management, but also in the adjuvant setting. J. Cell. Physiol. 226: 780–784, 2011. © 2010 Wiley‐Liss, Inc.     

Glycated Haemoglobin Is Inversely Related to Serum Vitamin D Levels in Type 2 Diabetic Patients

Objective

A correlation between glucose control and 25(OH)D metabolism has been suggested by previous studies. However, this correlation has not yet been evaluated considering the impact of chronic complications of type 2 diabetes, especially the presence of nephropathy. Thus, the aim of this study was to determine the correlation between A1C and 25(OH)D in a well characterized cohort of type 2 diabetic patients.

Research Design and Methods

We cross-sectionally examined the association between A1C and serum 25(OH) D in 715 type 2 diabetic patients attending our clinic during the years 2011–2012. The average age was 68±12 years (range 26–94 years). The relation between A1C and serum 25(OH)D levels was modelled by multiple linear regression analyses.

Results

Serum 25(OH)D levels were inversely associated with A1C levels (r = −0.116, p = .003). This relation maintains its independence in the multivariate analysis after adjusting for age, sex, A1C, BMI, treatment and duration of diabetes and nephropathy.

Conclusions

In type 2 diabetic patients, high A1C levels are associated with low concentrations of serum 25(OH)D independently of duration of diabetes, diabetic treatment and nephropathy. Future studies are needed to clarify the biological relation between glucose control and vitamin D metabolism in type 2 diabetes.     

Cholesterol-dependent modulation of the toxicity of HIV-1 coat protein gp120 in human neuroblastoma cells

The human immunodeficiency virus type 1 (HIV-1) coat glycoprotein gp120 binds to its (co)receptors and orchestrates cell entry by the direct fusion of viral and target cell membranes. Here, we modulated membrane fluidity of human neuroblastoma CHP100 cells by modulating their cholesterol content, and investigated the ability of gp120 to induce cell death in comparison with the untreated cells. We show that in normal CHP100 cells gp120 induces necrosis by: (i) increased cyclooxygenase and 5-lipoxygenase activity, and metabolites generated thereof (prostaglandin E2 and leukotriene B4, respectively); (ii) increased membrane lipoperoxidation; and (iii) increased mitochondrial uncoupling. These events were triggered by a rapid increase in intracellular calcium, and in cholesterol-depleted cells engaged CXCR4 chemokine receptors. The intracellular calcium chelator EGTA-AM protected CHP100 cells almost completely against the toxic effects of gp120. However, gp120-induced necrosis and related biochemical changes were negligible in cholesterol-enriched, and significantly enhanced in cholesterol-depleted, CHP100 cells exposed to the viral glycoprotein under the same experimental conditions. Taken together, these results suggest that membrane fluidity may control the neurotoxic effects of HIV-1 glycoprotein gp120.     

Exploiting RNA as a new biomolecular target for synthetic polyamines

Anticancer chemotherapy is strongly hampered by the low therapeutic index of most anticancer drugs and the development of chemoresistance. Therefore, there is a continued need for the identification of new molecular targets in order to selectively hit cancer cells. RNA has been recently validated as a cancer target by the use of different specific ligands and/or by different agents able to destroy its diverse forms. The ability of synthetic polyamines to interact and to alter the RNA structure has been already reported. In the present paper the interaction and the ability to damage RNA structure by several synthetic polyamines were evaluated and quantified by microfluid capillary electrophoresis. This technique allowed us to visualize both the RNA impairment through different electropherograms and to assess the RNA integrity number. Finally, the ability to discriminate between RNA and DNA by these synthetic polyamines was also evaluated.     

High avidity antigen-specific CTL identified by CD8-independent tetramer staining

Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.     

Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44, 276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 M hydrogen peroxide. Ascorbate (25 microM) or riboflavin (500 microM) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure.     

Ca2+-antagonists inhibit the N-methyltransferase‐dependent synthesis of phosphatidylcholine in the heart

Evidence indicates that, in addition to the Ltype Ca²⁺ channel blockade, Ca²⁺antagonists target other functions including the Ca²⁺pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca²⁺pumps by verapamil and diltiazem could be due to druginduced depression of phosphatidylethanolamine (PE) Nmethylation which modulates these Ca²⁺transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of SadenosylLmethionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10^–6–10^–3 M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10^–3 M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10^–5 M) was associated with a depression of the V_max value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE Nmethylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [³H-methyl]methionine following the treatment of animals with verapamil, reduced the synthesis of PC by Nmethyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca²⁺pump and although diltiazem also inhibited the SR Ca²⁺pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca²⁺-release. These results suggest that verapamil and diltiazem alter the cardiac PE Nmethyltransferase system. This action is apparently additional to the drugs' effect on Ltype Ca²⁺ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca²⁺pumps and altered cardiac function.