Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold

A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (V(H)H) as a crystallization chaperone. By using a yeast surface display system for V(H)H, we successfully introduced additional Met residues in the core of the V(H)H scaffold. We identified a set of SeMet-labeled V(H)H variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched V(H)H chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and V(H)H model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for V(H)H, the enhanced V(H)H domain described here will be an excellent scaffold for producing effective crystallization chaperones.     

Evaluating the diagnostic significance of nuclear grooves in thyroid fine needle aspirates with a semiquantitative approach

OBJECTIVE: To clarify the diagnostic significance of nuclear grooves in thyroid fine needle aspirates, to search for other associated nuclear features and to identify possible diagnostic pitfalls. STUDY DESIGN: In total, 67 fine needle aspiration cases were retrospectively reviewed for selected nuclear features, including grooves, intranuclear cytoplasmic invaginations, enlargement, pleomorphism and status of nucleoli. A semiquantitative method was used to estimate the percentage of nuclear grooves in 5 high-power fields with the most frequent nuclear grooves. RESULTS: Unequivocal intranuclear cytoplasmic invaginations were a specific diagnostic feature of papillary carcinoma. In the absence of intranuclear cytoplasmic invaginations, the presence of > or = 20% nuclear grooves was virtually diagnostic of thyroid neoplasms, predominantly papillary carcinoma. Less than 10% of nuclear grooves virtually eliminated the possibility of papillary carcinoma. However, 10-19% of nuclear grooves may be seen in papillary carcinoma and benign lesions; therefore, such cases fell into the diagnostic category of "atypical cytology" or "indeterminate" cytology. Nuclear enlargement with elongation was a readily identifiable feature usually associated with nuclear grooves. Significant pleomorphism was more commonly seen in papillary carcinoma than in benign lesions. CONCLUSION: A semiquantitative method is helpful when nuclear grooves are encountered in thyroid cytology in the absence of other diagnostic features. It can significantly improve the diagnostic specificity of nuclear grooves for papillary carcinoma.     

Effects of red wine consumption on serum paraoxonase/arylesterase activities and on lipoprotein oxidizability in healthy-men

Although there is a general consensus concerning the lower risk for cardiovascular disease in moderate drinkers, the mechanisms responsible for the cardioprotective effect of red wine remain unknown. It has been proposed that increased serum paraoxonase activity may be a mechanism of action underlying reduced cardiovascular disease risk in moderate drinkers, since paraoxonase inhibits lipoprotein oxidation. The aim of this study was to investigate the effects of red wine consumption on serum paraoxonase/arylesterase activities and on lipoprotein oxidizability in healthy-men. Fourteen healthy-men were included in the study. The subjects consumed 0.375 g alcohol / kg body weight for 3 weeks. Paraoxonase and arylesterase activities were studied spectrophotometrically. Oxidizability of apolipoprotein B-containing lipoproteins were determined, after separating them with precipitation method, by incubating with copper-sulfate. Paraoxonase activity did not change, however arylesterase activity significantly decreased after red wine consumption (P < 0.01). There was a reduced susceptibility of apolipoprotein B-containing lipoproteins to copper-sulfate induced oxidation after red wine consumption (P < 0.01). Our results support that red wine protects lipoproteins against oxidation, however there was not any significant change in serum paraoxonase activity after red wine consumption.KEY WORDS: Red wine; Paraoxonase; Arylesterase; Lipoprotein oxidation     

A novel and short synthesis of (1,4/2)-cyclohex-5-ene-triol and its conversion to (+/-)-proto-quercitol

(1,4/2)-cyclohex-5-ene-triol was synthesized starting from cyclohexa-1,4-diene with two different approaches. Photooxygenation of cyclohexa-1,4-diene and epoxy-cyclohexene afforded anti-2,3-dioxabicyclo[2.2.2]oct-7-en-5-yl hydroperoxide and anti-7-oxabicyclo[4.1.0]hept-4-en-3-yl hydroperoxide, respectively. Hydroperoxy endoperoxide was reduced with aqueous sodium bisulfite; hydroperoxy-epoxide with dimethylsulfide-titanium tetraisopropoxide to give 7-oxabicyclo[4.1.0]hept-4-en-3-ol. Acidic hydrolysis of the epoxy-alcohol gave the (1,4/2)-cyclohex-3-ene-triol. Oxidation of the double bond with KMnO4 resulted in the formation of (+/-)-proto-quercitol.     

Functional properties of threefold and fourfold channels in ferritin deduced from electrostatic calculations

The iron storage protein ferritin contains threefold and fourfold symmetric channels that are thought to provide pathways for the transfer of Fe(2+) ions in and out of the protein. Using the known crystal structure of the ferritin protein, we perform electrostatic potential energy calculations to elucidate the functional properties of these channels. The threefold channel is shown to be responsible for the transit of Fe(2+) ions. Monovalent ions can also diffuse through the threefold channel but presence of divalent ions in the pore retards this process leading to a selectivity mechanism similar to the one observed in calcium channels. The fourfold channel is found to be impermeant to all cations with the possible exception of protons. Because proton transfer is essential to maintain the electroneutrality of the protein during iron deposition, we suggest that the function of the fourfold channel is to form a "proton wire" that facilitates their transfer in and out of ferritin.     

Plasma soluble intercellular adhesion molecule 1 levels are increased in type 2 diabetic patients with nephropathy

BACKGROUND/AIM: Intercellular adhesion molecule 1 (ICAM-1) is a mediator in the recruitment of leukocytes in the glomerular cells. The role of ICAM-1 in diabetic complications is still a matter of debate. This study was performed to investigate the relation of plasma soluble ICAM-1 (sICAM-1) to nephropathy in patients with type 2 diabetes mellitus. METHODS: Ninety-three patients (24 males and 69 females) with type 2 diabetes mellitus were included into the study. Fifty patients had nephropathy, and 43 were free from nephropathy. Fifty healthy subjects (14 males and 36 females) served as the control group (group 1). Twenty-five of the diabetic patients had microalbuminuria (group 2), 25 had macroalbuminuria (group 3), and 43 had neither micro- nor macroalbuminuria (group 4). The plasma sICAM-1 levels were measured in blood samples drawn after fasting. RESULTS: The mean plasma sICAM-1 levels were not different in the 93 diabetic patients as compared with the healthy controls (392.7 +/- 119.5 vs. 350.1 +/- 90.2 ng/ml, p > 0.05). The mean sICAM-1 level was significantly higher in the diabetic patients with nephropathy than in those without nephropathy (430.3 +/- 78.2 vs. 368.2 +/- 122.5 ng/ml, p = 0.03) and in the controls (430.3 +/- 78.2 vs. 350.1 +/- 90.2 ng/ml, p = 0.016). The difference in sICAM-1 levels between groups 2 and 3 was not significant (p > 0.05). The plasma sICAM-1 levels were significantly higher in both groups 2 and 3 than in both groups 1 and 4 (434.5 +/- 129.2 vs. 427.2 +/- 113.7 ng/ml and 368.2 +/- 122.5 vs. 350.1 +/- 90.2 ng/ml, respectively). CONCLUSIONS: The plasma sICAM-1 levels in patients with type 2 diabetes mellitus are not significantly different from those in nondiabetic subjects. High levels of sICAM-1 suggest that sICAM-1 may play a role in the development of nephropathy in patients with type 2 diabetes mellitus.     

Effects of added dietary taurine on erythrocyte lipids and oxidative stress in rabbits fed a high cholesterol diet

Lipid peroxidation leads to damage of polyunsaturated fatty acids of membrane phospholipids. The contribution of oxidative stress to hypercholesterolemia-induced hemolytic anemia and the effects of addition of taurine on erythrocyte lipid composition, oxidative stress, and hematological data were studied in rabbits fed on a high cholesterol (HC) diet (1%, w/w) for 2 months. The effects of taurine on erythrocyte hemolysis and H2O2-induced lipid peroxidation were investigated in normal rabbit erythrocytes in vitro. The HC diet resulted in increases in plasma lipids and lipid peroxide levels as well as increases in cholesterol levels and the cholesterol:phospholipid ratio in the erythrocytes. This diet caused a hemolytic anemia, but lipid peroxide levels remained unchanged in the erythrocytes of the rabbits. Taurine (2.5%, w/w) added to the food has an ameliorating effect on plasma lipids and lipid peroxide levels in rabbits fed on a HC diet. This treatment also caused decreases in elevated erythrocyte cholesterol levels and cholesterol:phospholipid ratio due to the HC diet, but it did not prevent the hemolytic anemia and did not change erythrocyte lipid peroxide levels. In addition, in an in vitro study, taurine did not protect erythrocytes against H2O2-induced hemolysis or lipid peroxidation. These results show that the HC diet causes hemolytic anemia without any changes in erythrocyte lipid peroxidation, and taurine treatment was not effective against hemolytic anemia caused by the HC diet.