A Raman spectroscopic study of the interaction between nucleotides and the DNA binding protein gp32 of bacteriophage T4

Raman spectra of the bacteriophage T4 denaturing protein gp32, its complex with the polynucleotides poly(rA), poly(dA), poly(dT), poly(rU), and poly(rC), and with the oligonucleotides (dA)8 and (dA)2, were recorded and interpreted. According to an analysis of the gp32 spectra with the reference intensity profiles of Alix and co-workers [M. Berjot, L. Marx, and A.J.P. Alix (1985) J. Ramanspectrosc., submitted; A.J.P. Alix, M. Berjot, and J. Marx (1985) in Spectroscopy of Biological Molecules, A. J. P. Alix, L. Bernard, and M. Manfait, Eds., pp. 149-154], 1 gp32 contains ≈ 45% helix, ≈ 40% β-sheet, and 15% undefined structure. Aggregation of gp32 at concentrations higher than 40 mg/mL leads to a coordination of the phenolic OH groups of 4-6 tyrosines and of all the sulfhydryl (SH) groups present in the protein with the COO- groups of protein. The latter coordination persists even at concentrations as low as 1 mg/mL. In polynucleotide-protein complexes the nucleotide shields the 4-6 tyrosine residues from coordination by the COO- groups even at high protein concentration. The presence of the nucleotide causes no shielding of the SH groups. With Raman difference spectroscopy it is shown that binding of the protein to a single-stranded nucleotide involves both tyrosine and trytophan residues. A change in the secondary structure of the protein upon binding is observed. In the complex, gp32 contains more -sheet structure than when uncomplexed. A comparison of the spectra of complexed poly(rA) and poly(dA) with the spectra of their solution conformations at 15°C reveals that in both polynucleotides the phosphodiester vibration changes upon complex formation in the same way as upon a transition from a regular to a more disordered conformation. Distortion of the phosphate-sugar-base conformation occurs upon complex formation, so that the spectra of poly(rA) and poly(dA) are more alike in the complex than they are in the free polynucleotides. The decrease in intensity of the Raman bands at 1304 cm-1 in poly(rA), at 1230 cm-1 in poly(rU), and at 1240 and 1378 cm-1 of poly(dT) may be indicative of increased stacking interactions in the complex. No influence of the nucleotide chain length upon the Raman spectrum of gp322 in the complex was detected. Both the nucleotide lines and the protein lines in the spectrum of a complex are identical in poly(dA) and (dA)8

The MCGA (multiple cubic gradient approximation) method for the analysis of Raman spectra

An easily accessible interactive method for the analysis of Raman spectra consisting of many overlapping peaks is presented. A combination of a three- or four-dimensional grid and gradient searching is applied. The method can handle spectra with up to about 50 lines, based on a broad background. Analytical and user-defined or tabulated basic functions can be included. The merits of the method are discussed with both artificial and real spectra

Surface-enhanced Raman spectroscopy of DNA bases

A Raman microprobe has been used to measure the surface-enhanced Raman spectra of adenine, guanine, cytosine and thymine. Comparison of the SERS spectrum with solution spectra shows that some line positions are not influenced by the adsorption process while others show large shifts. In the SERS spectrum new lines, not visible in the solution spectrum, appear while some lines visible in the solution spectrum are not enhanced to a detectable level and are therefore not seen in SERS. The relative intensities are changed owing to an apparently vibration-dependent enhancement factor. A line-broadening effect occurs for most lines except carbonyl stretching vibrations in cytosine and thymine. All SERS spectra show increased contributions of bending vibrations and side-chain groups. In particular, amino group vibrations in adenine and cytosine are clearly visible. Comparison of the shape and intensity of the carbonyl stretching vibrations in cytosine, thymine and guanine show important differences. It is hypothesized that these differences indicate differences in the orientation of these groups with respect to the surface

Description and performance of a highly sensitive confocal Raman microspectrometer

A confocal Raman microspectrometer was developed for the study of small biological objects such as single living cells and metaphase and polytene chromosomes. It employs a confocal detection scheme, well known from confocal fluorescence microscopes, in order to avoid signal contributions from the environment of the samples. The resolution is 0.45 ± 0.05 m in the lateral direction and 1.3 ± 0.1 m in the axial direction. The laser excitation wavelength is 660 nm. At this wavelength biological samples do not degrade in the laser radiation as was the case when laser radiation of 514.5 nm was used. The signal throughput from the sample position to the detector was optimized to the extent that in the spectral region around a 1000 cm-1 Raman shift 15% of the Raman scattered light collected by the microscope objective is detected. For signal detection a liquid nitrogen-cooled slow-scan CCD camera is used. Laser powers of 5-10 mW suffice to obtain high-quality Raman spectra, with signal integration times of the order of minutes. As an example, spectra obtained from the nucleus and the cytoplasm of an intact human lymphocyte are shown

Two small-volume electrochemical cells for the measurement of surface enhanced Raman scattering

Two electrochemical cells, for performing surface enhanced Raman scattering (SERS), with submillilitre volumes are presented. One of the cells is especially developed for use in a Raman microspectrometer. The smallest cell uses only 80 mu l of sample. SER measurements are performed on 2*10-3 M adenine

Impacts of Rainfall and Forest Cover Change on Runoff in Small Catchments: A Case Study of Mulunguzi and Namadzi Catchment Areas in Southern Malawi

The impacts of climate change on water resources have received much attention globally especially in the last 30 years. Rainfall, the main driver of the hydrological cycle, has been varying in parts of the world in various ways. The picture is more complicated if impacts of land cover changes on water resources are also taken into consideration. These two pose challenges which require an integrated approach to address. Not many of such studies have been conducted in Malawian catchments and other tropical regions. In this study, annual, seasonal and monthly series of rainfall and river discharge of the Mulunguzi and Namadzi River catchments, two small sub-catchments in the Lake Chilwa catchment area, were analysed for trends using the non parametric Mann-Kendall statistic and Sens slope estimator. Further, Linear regression and the RainRU model were applied to establish whether the relationship between rainfall and runoff in the two catchments has changed. Furthermore, linear regression was used to establish how increased forest cover has influenced river flows in the two catchments. The results suggest that rainfall in the Mulunguzi catchment has decreased significantly at all scales and this has also led to reduced river flows. Increased forest cover since the pre 1950s has also resulted in reduced flows but this is not as significant as the rainfall decrease. In the Namadzi catchment, the rainfall trends suggest a varying pattern with no obvious straight trends. At annual and some months timescale, the rainfall has increased significantly. The river flow on the other hand suggests an overall declining pattern. This pattern is well linked with significant forest area increments which have occurred since 1995. It is therefore important that more detailed studies should be conducted to gain further insight to these relations as both catchments are important socioeconomically.Keywords: Land use; land cover; river flow; rainfall; climate change

Garlic reduces effect of Actinobacillus pleuropneumoniae infection in pigs

Lung diseases in pigs are among the most important health problem in pig husbandry, and generally treated with antimicrobials. To reduce the amount of antimicrobials used in pigs, we tested the preventive effects of freeze dried garlic added to feed on an infection with Actinobacilluspleuropneumoniae (APP). Thirty male pigs of about seven weeks of age were aerosol challenged with APP serotype 2. Fifteen pigs received 5% (w/w) garlic added to their diet (Garlic group), from two days prior to infection until four days after infection. The others received the a standard diet (Control group)

Perceptions on the use of pricing strategies to stimulate healthy eating among residents of deprived neighbourhoods: a focus group study

<p>Abstract</p> <p>Background</p> <p>Pricing strategies are mentioned frequently as a potentially effective tool to stimulate healthy eating, mainly for consumers with a low socio-economic status. Still, it is not known how these consumers perceive pricing strategies, which pricing strategies are favoured and what contextual factors are important in achieving the anticipated effects.</p> <p>Methods</p> <p>We conducted seven focus groups among 59 residents of deprived neighbourhoods in two large Dutch cities. The focus group topics were based on insights from Rogers' Diffusion of Innovations Theory and consisted of four parts: 1) discussion on factors in food selection; 2) attitudes and perceptions towards food prices; 3) thinking up pricing strategies; 4) attitudes and perceptions regarding nine pricing strategies that were nominated by experts in a former Delphi Study. Analyses were conducted with Atlas.ti 5.2 computer software, using the framework approach.</p> <p>Results</p> <p>Qualitative analyses revealed that this group of consumers consider price to be a core factor in food choice and that they experience financial barriers against buying certain foods. Price was also experienced as a proficient tool to stimulate healthier food choices. Yet, consumers indicated that significant effects could only be achieved by combining price with information and promotion techniques. In general, pricing strategies focusing on encouraging healthy eating were valued to be more helpful than pricing strategies which focused on discouraging unhealthy eating. Suggested high reward strategies were: reducing the price of healthier options of comparable products (e.g., whole meal bread) compared to unhealthier options (e.g., white bread); providing a healthy food discount card for low-income groups; and combining price discounts on healthier foods with other marketing techniques such as displaying cheap and healthy foods at the cash desk.</p> <p>Conclusion</p> <p>This focus group study provides important new insights regarding the use of pricing strategies to stimulate healthy eating. The observed perceptions and attitudes of residents of deprived neighbourhoods can be integrated into future experimental studies and be used to reveal if and how pricing strategies are effective in stimulating healthy eating.</p