Selective gamma-hydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/hypnotic effect of gamma-hydroxybutyric acid

Two gamma-hydroxybutyric acid (GHB) analogues, trans-gamma-hydroxycrotonic acid (t-HCA) and gamma-(p-methoxybenzyl)-gamma-hydroxybutyric acid (NCS-435) displaced [3H]GHB from GHB receptors with the same affinity as GHB but, unlike GHB, failed to displace [3H]baclofen from GABAB receptors. The effect of the GHB analogues, GHB and baclofen, on G protein activity and hippocampal extracellular glutamate levels was compared. While GHB and baclofen stimulated 5'-O-(3-[35S]thiotriphospate) [35S]GTPgammaS binding both in cortex homogenate and cortical slices, t-HCA and NCS-435 were ineffective up to 1 mm concentration. GHB and baclofen effect was suppressed by the GABAB antagonist CGP 35348 but not by the GHB receptor antagonist NCS-382. Perfused into rat hippocampus, 500 nm and 1 mm GHB increased and decreased extracellular glutamate levels, respectively. GHB stimulation was suppressed by NCS-382, while GHB inhibition by CGP 35348. t-HCA and NCS-435 (0.1-1000 microm) locally perfused into hippocampus increased extracellular glutamate; this effect was inhibited by NCS-382 (10 microm) but not by CGP 35348 (500 microm). The results indicate that GHB-induced G protein activation and reduction of glutamate levels are GABAB-mediated effects, while the increase of glutamate levels is a GHB-mediated effect. Neither t-HCA nor NCS-435 reproduced GHB sedative/hypnotic effect in mice, confirming that this effect is GABAB-mediated. The GHB analogues constitute important tools for understanding the physiological role of endogenous GHB and its receptor.     

Proteomics in Parkinson's disease: An unbiased approach towards peripheral biomarkers and new therapies

Parkinson's disease is the most common neurodegenerative movement disorder, affecting about 6 million people worldwide with a slow progression of the symptoms. Its prevalence is expected to double in the most populated areas within the next two decades, according to increasing aged population. Consequently, Parkinson's disease is a socio-economic trouble and a major challenge for the public health system. Parkinson's disease treatment is merely symptomatic, as clinical symptoms appear when about 70% of the involved neurons are lost and potential disease-modifying/neuroprotective therapies would have no effect. In turn, the availability of an objective measure that allows early diagnosis would strongly impact on the costs that biotech- and pharma-companies will sustain in order to develop disease-modifying therapies. The establishment of suitable models to investigate the mechanisms of Parkinson's disease progression and, on the other hand, the discovery and validation of selective and specific molecular biomarkers for early and differential diagnosis are indeed two important goals for a better management of the disease. In this review, we focus on cellular and animal models of Parkinson's disease by describing their advantages and limitations as useful tools to identify pathogenetic pathways that deserve further exploitation. In parallel, we discuss how proteomics may provide a potent tool to observe altered pathways in models or altered biomarkers in patients with an unbiased, hypothesis-free approach.     

The significance of an increase in soluble interleukin-2 receptor level in colorectal cancer and its biological regulating role in the physiological switching of the immune response cytokine network from TH1 to TH2 and back

 Current research has still not clarified the biological role of soluble interleukin(IL)-2 receptor (sIL-2R) and the significance of its increase in the serum of colon cancer patients compared to healthy subjects. To address these questions at the immunological level in a group of patients and healthy subjects, we determined the sIL-2R level in the serum and its release from peripheral blood mononuclear cells (PBMC) as a function of tumour necrosis factor (TNF) α, IL-1α, IL-1β, IL-2, interferon (IFN) γ, IL-4, IL-6 and IL-10 levels in the serum and PBMC production; and PBMC proliferative responses to IL-2, IL-4 and anti-CD3 monoclonal antibody (CD3), variously combined. The level of sIL-2R in patients’ serum was higher than in healthy subjects and correlated with the stage of advancement. Moreover, while in healthy subjects the serum level of sIL-2R was not significantly correlated with other parameters, in patients it was positively related to IL-4, IL-6 and IL-10 serum levels, PBMC IL-4 production and to the PBMC proliferative response to CD3 and CD3+IL-2; it was negatively correlated to IL-2 serum level and IL-1β PBMC release. A negative connection between IFNγ serum level and the PBMC production of sIL-2R was also found. This suggests that the increase of sIL-2R in the serum of patients, compared to healthy subjects, is involved in the inappropriate expansion of the T helper (TH2) suppressive immune response, which we previously reported. The multivariate statistical method supported the above suggestions and we also found that, in healthy subjects, the up- and down-regulation of sIL-2R in the serum within the physiological ranges seems to have a regulating role in the relationships between TNFα, IFNγ and IL-4, IL-6, contributing to the operation of the cytokine network between TH1 and TH2 cells. However, in patients compared to healthy subjects the increased sIL-2R serum level seems to direct the immune response towards a suppressive type, which may be due to an alteration in the above-mentioned physiological regulating role. Received: 12 April 1997 / Accepted: 4 September 1997     

SERS Properties of Gold Nanorods at Resonance with Molecular,Transverse, and Longitudinal Plasmon Excitations

The amplification of Raman signals of the heteroaromatic cation 1-(N-methylpyrid-4-yl)-2-(N-methylpyrrol-2-yl)ethylene (PEP+)) bound to Au nanorods (NRs) was investigated at different excitation wavelengths to study the effect of the laser resonance with the absorption band of the PEP+ moiety and with the two plasmon oscillation modes of the NR. Two different PEP+ derivatives, differing in the length of the alkyl chain bearing the anchoring group, were used as target molecules. Raman spectra obtained exciting at 514 or at 785 nm (i.e., exciting the transverse or the longitudinal plasmon band) present a higher intensity than that at 488 nm suggesting a higher Raman amplification when the laser excitation wavelength is resonant with one of the two plasmon modes. Moreover, considering results of Discrete Dipole Approximation (DDA) calculations of the local field generated at the NR surface when either the transverse or the longitudinal plasmon modes are excited, we deduced that the resonance condition of the 514-nm laser excitation with the absorption band of the dye strongly contributes to the amplification of the Raman signal.     

Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 μM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.     

β-thymosins and interstitial lung disease: study of a scleroderma cohort with a one-year follow-up

Background

β-thymosins play roles in cytoskeleton rearrangement, angiogenesis, fibrosis and reparative process, thus suggesting a possible involvement in the pathogenesis of systemic sclerosis. The aim of the study was to investigate the presence of thymosins β₄, β₄ sulfoxide, and β₁₀ in bronchoalveolar lavage fluid of scleroderma patients with interstitial lung disease and the relation of these factors with pulmonary functional and radiological parameters.

Methods

β-thymosins concentrations were determined by Reverse Phase-High Performance Liquid Chromatography-Electrospray-Mass Spectrometry in the bronchoalveolar lavage fluid of 46 scleroderma patients with lung involvement and of 15 controls.

Results

Thymosin β_4, β₄ sulfoxide, and β₁₀ were detectable in bronchoalveolar lavage fluid of patients and controls. Thymosin β₄ levels were significantly higher in scleroderma patients than in controls. In addition, analyzing the progression of scleroderma lung disease at one-year follow-up, we have found that higher thymosin β₄ levels seem to have a protective role against lung tissue damage. Thymosin β₄ sulfoxide levels were higher in the smokers and in the scleroderma patients with alveolitis.

Conclusions

We describe for the first time β-thymosins in bronchoalveolar lavage fluid and their possible involvement in the pathogenesis of scleroderma lung disease. Thymosin β₄ seems to have a protective role against lung tissue damage, while its oxidation product mirrors an alveolar inflammatory status.     

Cytokine expression and ultrastructural alterations in fresh-frozen,freeze-dried and γ-irradiated human amniotic membranes

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.