Handheld new technology Raman and portable FT-IR spectrometers as complementary tools for the in situ identification of organic materials in modern art.

A non-invasive approach has been carried out to characterize painting materials used in modern artworks conserved in the art collection of Carandente's museum at Palazzo Collicola in Spoleto (Italy). This work is focused on the cross-validation of the handheld BRAVO Raman spectrometer, that uses a sequentially Shifted Excitation (SSE) to mitigate fluorescence, for the characterization specifically of organic materials. The analytical procedure, combining XRF, Raman and reflection infrared spectroscopy, allowed a complete characterization of the artists' palettes; particularly eight different synthetic dyes belonging to the class of pigment red (PR) and pigment yellow (PY.), synthetic and traditional binders, such as alkyd resin and lipids have been easily identified.

Surface electrostatic interactions contribute little of stability of barnase.

Electrostatic interactions are believed to play an important role in stabilizing the native structure of proteins. We have quantified the contribution to stability of an interaction between two oppositely charged side-chains on the surface of barnase. Using site-directed mutagenesis, glutamate 28 and lysine 32 were introduced onto the solvent-accessible side of the second alpha-helix in barnase. These two residues are separated by one turn of the helix, and so are ideally situated for their opposite charges to interact. Double mutant cycle analysis reveals that the interaction between Glu28 and Lys32 contributes only approximately 0.2 kcal/mol to stability of the protein. All other interactions between exposed charged side-chains in barnase examined so far also contribute little to stability. We explain this low value by their location on the surface, rather than in the interior, of the protein.

Mortality of short-term workers in two international cohorts.

The purpose of this study was to compare the pattern of mortality of blue-collar workers employed less and more than 1 year in the man-made vitreous fiber (MMVF) and the reinforced plastic industries, the latter group being exposed to styrene. We conducted an analysis among 21,784 workers with less than 1 year of employment (short-term workers) and 19,117 workers with 1 or more years of employment (long-term workers) employed in eight European countries. We conducted analyses based on external as well as internal comparisons. In both cohorts, the standardized mortality ratio for all causes among short-term workers was approximately 40% higher, compared with that for longer-term workers. In internal comparisons, the difference was reduced to 9% in the MMVF cohort and 11% in the styrene cohort. Workers with less than 1 month of employment displayed an increased mortality in both cohorts and in most countries. The increased mortality among short-term workers was not concentrated shortly after they quit employment. In both cohorts, short-term workers had a higher mortality from external causes, while little difference was seen in mortality from ischemic heart disease and malignant neoplasms. Although extra-occupational factors may contribute to increase the mortality of short-term workers and, in particular, of those employed for less than 1 month, the difference observed in analyses adjusted for characteristics of employment suggested a relatively small difference in mortality from most causes.

Kidney cancer and occupational exposure to asbestos: a meta-analysis of occupational cohort studies.

OBJECTIVE: To study the risk of kidney cancer following asbestos exposure. METHODS: We carried out a meta-analysis of the results of cohort studies of workers predominantly exposed to asbestos. We contacted the authors of 70 cohort studies; published results were available from the reports of 10 cohorts; we obtained the relevant information for an additional 27 cohorts. RESULTS: The studies included in the analysis comprised a total of 169 kidney cancer deaths and 69 incident cases. The overall pooled standardized mortality ratio (SMR) of kidney cancer was 1.1, with a 95% confidence interval (95% CI) of 0.9-1.3. The pooled SMR was higher for workers with undefined asbestos exposure (SMR 1.2, 95% CI 0.9-1.6) than for workers with either predominant chrysotile (SMR 0.9, 95% CI 0.7-1.3) or some amphibole (SMR 0.96, 95% CI 0.6-1.5) exposure. Studies with published results had higher SMRs than studies for which information was obtained from the authors. Studies with high asbestos exposure and an elevated SMR of lung cancer tended to show an increased risk of kidney cancer. CONCLUSIONS: It is unlikely that asbestos exposure is responsible for an important increase in kidney cancer risk; however, high asbestos exposure might entail a small increase in risk.

Stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain.

The alpha-helix in proteins has a dipole moment resulting from the alignment of dipoles of the peptide bond which can perturb the pKas of ionizing groups. One of the two histidine residues (His18) in barnase, the small ribonuclease from Bacillus amyloliquefaciens, is located at the negatively charged end (C-terminal) of an alpha-helix. From NMR titrations of wild-type and engineered mutants we find that the pKa of His18 is 7.9 in wild-type enzyme, 1.6 units above the value in the urea-denatured enzyme and in model peptides. This implies that there is a favourable interaction between the protonated form of His18 and the alpha-helix that should stabilize the native structure at neutral pH by 2.1 kcal mol-1. Denaturation at various values of pH of wild-type and muant enzymes engineered at position 18 shows that this is so. The increase in stability of the enzyme as the pH changes from 8.5 to 6.3 is attributable to this interaction, and the pH-stability curve fits pKa values for His18 in native and urea-denatured enzymes that are consistent with the NMR data.

Contribution of hydrophobic interactions to protein stability.

A major factor in the folding of proteins is the burying of hydrophobic side chains. A specific example is the packing of alpha-helices on beta-sheets by interdigitation of nonpolar side chains. The contributions of these interactions to the energetics of protein stability may be measured by simple protein engineering experiments. We have used site-directed mutagenesis to truncate hydrophobic side chains at an alpha-helix/beta-sheet interface in the small ribonuclease from Bacillus amyloliquefaciens (barnase). The decreases in stability of the mutant proteins were measured by their susceptibility to urea denaturation. Creation of a cavity the size of a -CH2-group destabilizes the enzyme by 1.1 kcal mol-1, and a cavity the size of three such groups by 4.0 kcal mol-1.

Solution structure of a brodimoprim analogue in its complex with Lactobacillus casei dihydrofolate reductase.

Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1:1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-O position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of 10(3) more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 11 intramolecular NOEs were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tau 1 = -153 degrees +/- 4 degrees (C4-C5-C7-C1') and tau 2 = 53 degrees +/- 4 degrees (C5-C7-C1'-C2'): the aromatic rings of the ligand occupied essentially the same space in all the calculated structures (root mean square deviation value 1.83 A). Inclusion of the electrostatic interactions into the energy minimizations indicated that structures in which the 4,6-dicarboxylate group of the ligand interacts with the side chains of Arg 57 and His 28 are of low energy. Significant differences in side-chain and backbone conformations were detected between binding-site residues in the enzyme complexes with the brodimorpim analogue and methotrexate.

Non-neoplastic mortality of European workers who produce man made vitreous fibres.

OBJECTIVE: To study mortality from non-neoplastic diseases among European workers who produce man made vitreous fibres (MMVF). METHODS: 11,373 male workers were studied, who were employed for at least 1 year in the production of rock or slag wool (RSW), glass wool (GW), and continuous filament (CF) in 13 factories from seven European countries. Workers were followed up from the beginning of production, between 1933 and 1950 to 1990-2 and contributed 256,352 person-years of observation. Standardised mortality ratios (SMRs) were calculated with national mortalities for reference; an internal exposure-response analyses based on multivariate Poisson regression models was also conducted. RESULTS: Mortality from bronchitis, emphysema, and asthma was not increased (SMR 1.03, 95% confidence interval (95% CI) 0.82 to 1.28). In RSW workers, there was no overall increase in mortality from non-malignant renal diseases (SMR 0.97, 95% CI 0.36 to 2.11), although there was the suggestion of an increase in risk with duration of employment. Mortality from ischaemic heart disease was not increased overall (SMR 1.03, 95% CI 0.96 to 1.11), but RSW and CF workers with > or = 30 years since first employment had a higher risk. RSW and CF workers showed an increased mortality from external causes, mainly motor vehicle accidents and suicide, which was higher among workers with a short duration of employment. CONCLUSIONS: Mortality from most non-neoplastic diseases does not seem to be related to employment in the MMVF industry. The results on mortality from ischaemic heart disease and non-malignant renal diseases, however, warrant further investigations.

Update of the follow-up of mortality and cancer incidence among European workers employed in the vinyl chloride industry.

Although vinyl chloride is an established cause of liver angiosarcoma, the evidence is inconclusive on whether it also causes other neoplastic and nonneoplastic chronic liver diseases as well as neoplasms in other organs. Furthermore, the shape of the dose-response relation for angiosarcoma is uncertain. We have extended for approximately 8 years the mortality and cancer incidence follow-up of 12,700 male workers in the vinyl chloride industry in four European countries. All-cause mortality was lower than expected, whereas cancer mortality was close to expected. A total of 53 deaths from primary liver cancer (standardized mortality ratio 2.40, 95% confidence interval = 1.80-3.14) and 18 incident cases of liver cancer were identified, including 37 angiosarcomas, 10 hepatocellular carcinomas, and 24 liver cancers of other and unknown histology. In Poisson regression analyses we observed a marked exposure response for all liver cancers, angiosarcoma, and hepatocellular carcinoma. The exposure-response trend estimated for liver cancer in analyses restricted to cohort members with cumulative exposures of <1,500 parts per million-years was close to that estimated for the full cohort (relative risk of 2.0 per logarithmic unit of cumulative dose). No strong relation was observed between cumulative vinyl chloride exposure and other cancers. Although cirrhosis mortality was decreased overall, there was a trend with cumulative exposure.

Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex.

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein into its constituent nonstructural proteins. The NS3/4A complex is thus an attractive target for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during expression, producing a noncovalent complex between HisNS3 and NS4A with a subnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatography. We removed the His tag by thrombin cleavage and then further purified the complex by gel filtration. The purified NS3/4A complex is active in a protease assay using a synthetic peptide substrate derived from the NS5A-NS5B junction, with kcat/K(m) of 3700 (+/- 600) M-1 s-1, an order of magnitude above those previously reported for NS3 expressed by other strategies. This high protease activity implies that the full-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activity is dependent on the aggregation state of the NS3/4A complex. The monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.

Cancer consequences of the Chernobyl accident in Europe outside the former USSR: a review.

The accident which occurred during the night of April 25-26, 1986 in reactor 4 of the Chernobyl nuclear power plant in the Ukraine released considerable amounts of radioactive substances into the environment. Outside the former USSR, the highest levels of contamination were recorded in Bulgaria, Austria, Greece and Romania, followed by other countries of Central, Southeast and Northern Europe. Studies of the health consequences of the accident have been carried out in these countries, as well as in other countries in Europe. This report presents the results of a critical review of cancer studies of the exposed population in Europe, carried out on the occasion of the 10th anniversary of the Chernobyl accident. Overall, three is no evidence to date of a major public health impact of the Chernobyl accident in the field of cancer in countries of Europe outside the former USSR.