Serum Amyloid A Complexed with Extracellular Matrix Induces the Secretion of Tumor Necrosis Factor-α by human T-lymphocytes

Summary Serum amyloid A (SAA), an acute-phase reactant, exists naturally as a minor protein in the sera of healthy individuals. However, its levels in sera are increased markedly during various transient and chronic inflammatory diseases, often concomitantly with accumulation at inflicted sites. SAA is synthesized mainly in the liver following the synergistic action of cytokines, mainly tumor necrosis factor-α (TNF-α) and interleukin-1 and-6 (IL-1 and IL-6). It was already shown by us that upon interaction with SAA or amyloid A (AA), the extracellular matrix (ECM) and laminin induced the adhesion of resting human CD4⁺ T-cells in an apparently β₁-integrin-mediated manner. Herein we have shown that the SAA-ECM complex modulates the regulation of cytokine synthesis by human T-lymphocytes. The SAA-ECM complex dramatically enhanced the release of TNF-α by human T-cells in a dose-dependent manner, reaching its maximal effect in the presence of 100 μM recombinant SAA. The SAA domain, responsible for the enhanced release of TNF-α by human T-lymphocytes, is apparently the amyloid A protein (AA, i.e. SAA2-82). Specifically, TNF-α enhanced secretion is mediated through intimate interactions of SAA/AA, with laminin. Thus, the ECM serving as a temporary anchorage site for SAA and AA seems to be involved in regulating TNF-α secretion and the recruitment and accumulation of immunocytes in extravascular, inflammatory compartments.     

Cloning and Expression Analysis of Human Amelogenin in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> Plants by Means of a Transient Expression System

Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons. Both genes yielded approximately 500 µg of total amelogenin per gram of fresh leaf tissue. Two purification procedures based on affinity chromatography or on intrinsic solubility properties of the protein were followed, yielding from 12 to 150 µg of amelogenin per gram of fresh leaf tissue, respectively, at different purity. The identity of the plant-made human amelogenin was confirmed by MALDI-TOF–MS analysis of peptides generated following chymotrypsin digestion. Using dynamic light scattering, we showed that plant extracts made in acetic acid containing human amelogenin have a bimodal distribution of agglomerates, with hydrodynamic diameters of 22.8 ± 3.8 and 389.5 ± 86.6 nm. To the best of our knowledge, this is the first report of expression of human amelogenin in plants, offering the possibility to use this plant-made protein for nanotechnological applications.     

Cadmium affects the redox state of rat liver glucocorticoid receptor

It has previously been documented that cadmium displays high affinity for protein thiol groups and induces an impairment of glucocorticoid receptor (GR) cellular functions. The present study examined the possibility that cadmium exerts these effects on GR activity by disturbing the receptor's redox equillibrium. To that end, the influence of cadmium on the rat liver GR potential to form intramolecular and intermolecular disulfide bonds under nonreducing conditions and under oxidizing conditions produced by the addition of hydrogen peroxide (H₂O₂) to the cytosol was examined by nonreducing SDS-PAGE and immunoblotting. The results show that cadmium inhibits formation of disulfide bonds within the GR both in the absence and in the presence of H₂O₂. The creation of intermolecular disulfide linkages between the apo-GR and associated heat shock proteins Hsp90 and Hsp70, which was evident in the presence of H₂O₂, was also significantly impaired after cadmium administration. These observations are consistent with the assumption that cadmium affects the redox state of the receptor, possibly by binding to its sulfhydryl groups. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ki-67 and AgNOR proliferative markers as diagnostic adjuncts to fine needle aspiration cytology of thyroid follicular lesions

OBJECTIVE: To differentiate hyperplastic nodules (HPN), follicular adenoma (FA) and follicular carcinoma (FCA) of the thyroid by cytomorphologic features combined with argyrophilic nucleolar organizer regions (AgNORs) and Ki-67 proliferative markers on fine needle aspiration cytology. STUDY DESIGN: Cytomorphologic patterns, along with two proliferation markers, Ki-67 and AgNORs, in fine needle aspirates of 123 histologically confirmed cases of thyroid follicular lesions, including 39 hyperplastic nodules, 70 follicular adenomas and 14 cases of follicular carcinomas, were recorded. RESULTS: Mean AgNOR (mAgNOR) counts and Ki-67 labelling index (LI) were consistently higher in FCA in comparison to FA and HPN irrespective of the cytologic patterns in fine needle aspiration smears. Between benign and malignant lesions, an overlap of 1.83% at the cutoff point of 4.0 was observed in cases of mAgNORs, whereas it was 11.09% at a cutoff of 5.0 in cases of Ki-67 LI. CONCLUSION: mAgNOR counting in fine needle aspiration smears is more sensitive, simple and cost effective as compared to Ki-67 LI for differentiating between benign and malignant thyroid follicular neoplasms.     

Six-membered cyclic ureas as HIV-1 protease inhibitors: a QSAR study based on CODESSA PRO approach. Quantitative structure-activity relationships

Quantitative structure-activity relationships (QSAR) for HIV-1 protease inhibitory activity of substituted tetrahydropyrimidinones have been produced using CODESSA PRO methodology and software. The best four-parameter equation (R(2)(cv)=0.847) allowed us to reveal two main structural factors which are strongly correlated with the title activity: molecular hydrophobicity and ability to form hydrogen bonds with the target enzyme.     

Mercury inhibits rat liver and kidney glucocorticoid receptor hormone binding activity

The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined after in vivo and in vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when applied in vitro at 0 degrees C, mercury produced reduction of the receptor binding activity similar to that observed in vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.