Insight into the stability of the hydrophobic binding proteins of Escherichia coli: assessing the proteins for use as biosensors

Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins. Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model. Ligand binding causes significant stabilization of both proteins. There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein. A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors.     

Chemisorptions of bacterial receptors for hydrophobic amino acids and sugars on gold for biosensor applications: a surface plasmon resonance study of genetically engineered proteins

This paper demonstrates potential applications of two periplasmic receptor proteins from E. coli as sensing elements for biosensors using the surface plasmon resonance (SPR) technique. These molecules, namely the aspartate to cysteine mutant of the leucine-specific receptor (LS-D1C) and the glutamine to cysteine mutant of the D-glucose/D-galactose receptor (GGR-Q26C) proteins, are chemisorbed on a thin (approximately 40 nm) Au film in neutral K2HPO4 buffers. Using angle and time resolved SPR measurements; we show that adsorption behaviors of both proteins are dominated by diffusion-free second order Langmuir kinetics. We also show that the protein-modified Au films exhibit measurable SPR shifts upon binding to their respective target ligands. According to these SPR data, the kinetics of ligand binding for both LS-D1C and GGR-Q26C are governed by irreversible first order diffusion limited Langmuir model. The utility of the SPR technique for studying reactions of biological molecules is further illustrated in this work.     

Comparative study of lignins isolated from Alfa grass (Stipa tenacissima L.)

Soda lignin, dioxane lignin and milled lignin were isolated from Alfa grass (Stipatenacissima L.). The physico-chemical characterization of three different lignins: one industrial lignin precipitated from soda spent liquor and two lignin preparations isolated under laboratory conditions from Alfa grass (also know as Esparto grass) was performed. The structures of lignins were studied by three non-destructive (FT-IR, solid state ¹³C NMR and UV/visible spectroscopy) and two destructive (nitrobenzene oxidation and thermogravimetric analysis) methods. Elemental analysis and the methoxyl content determination were performed in order to determine the C₉ formulae for the studied lignins. The total antioxidant capacity of the studied lignins has been determined and compared to commercial antioxidants commonly used in thermoplastic industry.     

Comparative study of the genotoxic response of freshwater mussels Unio tumidus and Unio pictorum to environmental stress

Genotoxic response of freshwater mussels U. tumidus and U. pictorum to environmental stress was studied using comet assay on hemocytes and gill cells. The mussels were acclimated to controlled laboratory conditions for 10 days, and then exposed at 4 sites in the Sava and Danube rivers in the area of the city of Belgrade. Samples of each species were taken after 7, 14, and 30 days of exposure. The mussels sampled immediately after acclimation served as controls. Genotoxic response in both species was induced earlier at sites receiving untreated wastewaters from the city’s main collectors (7 days), than at the site receiving only domestic wastewaters from small settlements located upstream from the city (30 days). There was a correlation between the comet tail intensity values in tissues of exposed mussels and the concentrations of zinc, copper, iron, and arsenic at the exposure sites. The genotoxic responses in both tissues of U. pictorum and in hemocytes of U. tumidus were similar, while U. tumidus gill cells failed to exhibit significant genotoxic response at two sites. These findings, together with higher mortality of U. tumidus at the most polluted sites, promote U. pictorum as a model for genotoxicity monitoring in freshwater environments.     

Free radical–scavenging activity and DNA damaging potential of auxins IAA and 2‐methyl‐IAA evaluated in human neutrophils by the alkaline comet assay

Auxins, of which indole‐3‐acetic acid (IAA) is the most widespread representative, are plant hormones. In addition to plants, IAA also naturally occurs in humans in micromolar concentrations. In the presence of peroxidase, indolic auxins are converted to cytotoxic oxidation products and have thus been proposed for use in gene‐directed enzyme/prodrug tumor therapy. Since data on the genotoxicity of IAA and its derivatives are not consistent, here we investigate the early DNA damaging effects (2‐h treatment) of the auxins, IAA, and 2‐methyl‐indole‐3‐acetic acid (2‐Me‐IAA) by the alkaline comet assay and compare them with their free radical–scavenging activity measured by the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay. Human neutrophils are chosen as the test system since they possess inherent peroxidase activity. The results of the comet assay indicate an increase in DNA damage in a dose‐dependent manner up to 1.00 mM of both auxins. Generally, IAA applied in the same concentration had greater potential to damage DNA in human neutrophils than did 2‐Me‐IAA. The genotoxicities of the two examined auxins are negatively correlated with their antioxidant activities, as measured by the DPPH assay; 2‐Me‐IAA showed a higher antioxidant capacity than did IAA. We assume that differences in the molecular structure of the tested auxins contributed to differences in their metabolism, in particular, with respect to interactions with peroxidases and other oxidative enzymes in neutrophils. However, the exact mechanisms have to be elucidated in future studies. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:165–173, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20323     

Evaluation of genotoxic effects of fumagillin by cytogenetic tests in vivo

Fumagillin is a naturally secreted antibiotic of the fungus Aspergillus fumigatus. It is used in veterinary medicine against microsporidiosis of bees and fish. In this study, the genotoxicity of fumagillin (in the form of fumagillin dicyclohexylamine) was evaluated in mouse bone-marrow cells using the mitotic index (MI), the chromosome aberration (CA) assay, and the micronucleus (MN) test. Fumagillin was administered to BALB/c mice by gavage, at doses of 25, 50, 75 mg/kg body weight (bw), repeated for 7 days at 24-h intervals, with water-sugar syrup as a negative control and cyclophosphamide (40 mg/kg bw) as a positive control. All experimental doses of fumagillin induced a significant decrease (p<0.001) in MI (3.47+/-0.04%, 3.17+/-0.01%, and 2.27+/-0.02%, respectively) in comparison with the negative control (6.00+/-0.01%). Fumagillin significantly (p<0.001) increased the frequency of MN (4.98+/-0.35, 8.45+/-0.57, and 12.02+/-0.37, respectively) over negative control (1.04+/-0.28). Significantly increased frequencies (p<0.01 or p<0.001) of numerical chromosomal aberrations (aneuploidies and polyploidies) and structural chromosomal aberrations such as gaps, breaks, and centric rings were observed at the highest experimental dose of fumagillin (75 mg/kg bw) compared with the negative control. However, with respect to the induction of Robertsonian translocations, both the intermediate (50 mg/kg bw) and highest (75 mg/kg bw) experimental dose caused a significant (p<0.001) increase (7.12+/-0.26 and 9.00+/-0.10, respectively) in comparison with the negative control (0.00+/-0.00). Chromosomes 4 and 19 participated in these Robertsonian translocations. Regarding total cytogenetic changes, a significant increase (p<0.001) was observed in both the intermediate dose group (17.36+/-1.83) and the highest dose group (59.49+/-1.92) compared with the negative control (7.00+/-1.35). These results suggest that fumagillin has genotoxic (clastogenic) potential in mammals in vivo.     

A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin

Hemocyanins of mollusks are high molecular mass glycoproteins with a complex quaternary structure which still remains to be defined in detail for most of its species as far as number, spatial distribution and interactions of their structural units is concerned. In the present study, we isolated the functional units of the structural subunit RvH1 of Rapana venosa hemocyanin, combining enzymatic and non-enzymatic methods. Our results suggest that Hc's carbohydrate moieties play a basic role in the organization of the structural units, resulting from post-translational polymerization of the 50 kDa functional units and involving sugar moieties that link between them.     

Assessment of CD4<Superscript>+</Superscript> T cells specific for the tumor antigen SSX-1 in cancer-free individuals

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.     

A single-centre experience with octreotide in the treatment of different hypersecretory syndromes in patients with functional gastroenteropancreatic neuroendocrine tumors

The aim of this research was to assess the clinical and biochemical efficacy of the octreotide in the treatment of patients with various functional gastroenteropancreatic neuroendocrine tumors (GEP-NETs). The study included 14 patients treated with octreotide for 6 months. They were diagnosed with VIPoma, glucagonoma, gastrinoma, medullary thyroid carcinoma (solitary and as a part of MEN-II syndrome), pancreatic carcinoids (solitary and as a part of multiple endocrine neoplasia type-1 syndrome-MEN-1 syndrome) and midgut carcinoids. The patients presented with Verner-Morrison, glucagonoma, Zollinger Ellison and carcinoid syndrome respectively. All had a metastatic disease at the time of diagnosis and a positive octreoscan finding. Initially elevated chromogranin A (CgA) levels were detected in 11 (78.6%) and elevated 5-hydroxyindolacetic acid (5-HIAA) levels in 8 (57.1%) patients. Symptomatic efficacy assessments were made by diarrhea reductions during treatment course, and laboratory efficacy was assessed through changes in 5-HIAA and CgA levels. Assessments were made initially and following 6 months of therapy. Median urinary 5-HIAA and the number of stools decreased significantly (p = 0.016 and p = 0.009 respectively, p < 0.05) while CgA levels had the decreasing tendency but not statistically significant (p = 0.14). There was a positive correlation between the 5-HIAA reduction and the decrease in stool number at baseline and during treatment course (p < 0.05). No correlation was observed between 5-HIAA and CgA levels and also there was no correlation between CgA reduction and symptomatic improvement. The results prove octreotide to be effective in reducing symptoms and biochemical markers associated with hypersecretory syndromes of GEP-NETs.