Surface-enhanced Raman spectroscopy analysis of house paint and wallpaper samples from an 18th century historic property.

Conservation efforts for heritage buildings require a substantial knowledge of the chemical makeup of materials that were used throughout the lifetime of the property. In particular, conservators are often concerned with the identification of colorants used in both interior and exterior wall treatments (paint, wallpaper, etc.) in order to gain perspective into how the building may have appeared during a certain time period in its existence. Ideally, such an analysis requires a technique that provides molecular level information as to the identity of the colorant as well as other sample components (binders, fillers, etc.), which is useful for dating purposes. In addition, the technique should be easily applied to paint layer samples which can be extremely thin and fragile. Herein we report the surface-enhanced Raman spectroscopy (SERS) analysis of paint and wallpaper samples taken from exterior and interior surfaces of a historic building. Several pigments were identified in the samples, which ranged from early inorganic pigments (lead white, barium sulfate, calcium carbonate, anhydrous chromium(III) oxide) which have been used in house paints for centuries, to a more modern pigment (phthalocyanine blue), developed in the middle of the 20th century. This analysis highlights the usefulness of SERS in such a conservation effort, and demonstrates for the first time pigment identification in house paints and wallpaper using SERS, which has far-reaching implications not only in the field of conservation, but also in forensics, industrial process control, and environmental health and safety.

Evaluation of pet food by-product as an alternative feedstuff in weanling pig diets.

Three experiments were conducted to evaluate pet food by-product (PFB) as a component of nursery starter diets and its effects on pig performance. The PFB used in these studies was a pelleted dog food that contained (as-fed basis) 21% CP, 1.25% total lysine, and 8.3% ether extract. In Exp. 1, 288 early-weaned pigs (5.2 kg at 14 d) were used to determine the effects of replacing animal protein and energy sources with PFB at 0, 10, 30, and 50% (as-fed basis) inclusion levels in phase I (d 0 to 7 after weaning) and phase II (d 7 to 21 after weaning) diets. Phase I diets contained 27.5% whey, 18.75% soybean meal, 1.50% lysine, 0.90% Ca, and 0.80% P, with PFB substituted for corn, fat, plasma protein, fish meal, limestone, and dicalcium phosphate. Phase II diets had a constant 10% whey, 1.35% lysine, and PFB was substituted for blood cells, a portion of the soybean meal, and other ingredients as in phase I diets. In phase I, growth performance by pigs fed PFB-containing diets was similar to that of the control diet. In phase II, ADG (linear; P < 0.05 and quadratic, P < 0.005), ADFI (linear and quadratic, P < 0.01), and G:F (quadratic, P < 0.01) were increased with increasing PFB inclusion. In Exp. 2, 80 weaned pigs (6.7 kg at 21 d) were fed a common phase I diet for 1 wk and used to further evaluate the effect of PFB in phase II diets (same as Exp 1; initial BW = 8.1 kg) on growth performance and apparent total tract nutrient digestibility. There were no differences in ADG, ADFI, or G:F across treatments. Dry matter and energy digestibility did not differ among diets; however, digestibilities of CP (P < 0.05) and the essential AA, arginine (P < 0.02), histidine (P < 0.01), lysine (P < 0.001), threonine (P < 0.01), and valine (P < 0.01), were greater as PFB was increased in the diet. In Exp. 3, the performance by pigs (n = 1 70; 5.5 kg; 21 d of age) fed diets with 0 or 30% PFB in both phases I and II was examined. Growth performance was similar in both diets. These studies demonstrate that pet food by-product can effectively be used as a partial replacement for animal protein sources and grain energy sources in the diets of young nursery pigs.

Carbohydrate, glycopeptide and protein components of the lichen Sticta sp. and effect of storage.

Three aldoses, six alditols, eight oligosaccharides, three polysaccharides and a glycopeptide were found in a Sticta sp. The most interesting of these were gentiobiose, -triose and -tetraose, O-alpha-D-Galp-(1-->6)-O-beta-D-Galp-(1-->1)-D-glyceritol released on alkaline treatment, and a polysaccharide with predominant (1-->4)-linked beta-D-Xylp units. Present were a galactoglucomannan, a polysaccharide, possibly a mixture, containing beta-D-Galf, and alpha-L-Araf units and a glycopeptide with alpha- and beta-D-Galf, Galp, Glcp and Manp units, having a core with successive (1-->2)-linked alpha-D-Manp residues substituted at O-3 with those of alpha-D-Galf. After four years storage of the lichen, the contents of the principal polyols, ribitol (0.86%), arabinitol (0.99%) and mannitol (2.50%) fell to practically zero, with the appearance of threitol (0.03%), xylitol (0.01%) and myo-inositol (0.03%). Also, the protein content fell from 34 to 18%. The most abundant amino acids were valine (10.5% of total) in fresh lichen and threonine (14.6%) in stored lichen.

Comparison of solution hybridization efficiencies using alkaline phosphatase-labelled and 32P-labelled oligodeoxynucleotide probes.

The hybridization efficiencies of oligonucleotide probes directly labelled with alkaline phosphatase and probes labelled with 32P were compared by quantitating the enzyme activity or radioactivity associated with hybridization targets over time. The targets tested included both synthetic oligonucleotides (53 bases in length) and single-stranded and double-stranded cloned M13 DNA (7350 bases long). Hybrid molecules were separated from unhybridized probes using size exclusion FPLC. This system allowed quantitative analysis of the time course and efficiency of hybridization for both probes and targets in complex hybridization media containing protein blocking agents, formamide, and carrier DNA. Similar maximum hybridization efficiencies were attained for probes labelled with either radioactivity or alkaline phosphatase as a marker. The reaction rate constant for oligonucleotide hybridization to long M13 targets was 3.6 x 10(5) mol-1 s-1 for a probe labelled with alkaline phosphatase, and 5.8 x 10(5) mol-1 s-1 for the same probe labelled with 32P.

Use of enzyme-linked synthetic DNA in diagnosis of falciparum malaria.

Plasmodium falciparum contains a family of 21-base-long repetitive DNA sequences in its genome. A 21-base synthetic DNA oligomer, formerly labelled with phosphorus-32 for autoradiographic detection of P falciparum DNA, was covalently coupled to alkaline phosphatase for histochemical detection. The conjugate (PFR1-AP) detected purified P falciparum DNA with a sensitivity and specificity equal to that of 32P-labelled probes after 2-day exposures. PFR1-AP did not detect host DNA or DNA of other Plasmodium species. In African blood specimens PFR1-AP specifically detected P falciparum infections of 100 parasites/microliter. This sensitive, rapid, nonisotopic probe will allow more widespread use of DNA hybridisation in the diagnosis of malaria.

Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes.

Short synthetic oligonucleotides have been covalently cross-linked to alkaline phosphatase using the homobifunctional reagent disuccinimidyl suberate. The oligomers, twenty-one to twenty-six bases in length, are complementary to unique sequences found in herpes simplex virus, hepatitis B virus, Campylobacter jejuni and enterotoxigenic Escherichia coli. Each oligomer contains a single modified base with a 12-atom "linker arm" terminating in a reactive primary amine. Cross-linking through this amine results in oligomer-enzyme conjugates composed of one oligomer per enzyme molecule that have full alkaline phosphatase activity and can hybridize to target DNA fixed to nitrocellulose within 15 minutes. The hybrids are detected directly with a dye precipitation assay at a sensitivity of 10(6) molecules (2 X 10(-18) mol) of target DNA in 4 hours development time. The enzyme has no apparent effect on selectivity or kinetics of oligonucleotide hybridization and the conjugates can be hybridized and melted off in a conventional manner.

The preparation of bacterial luciferase conjugates for immunoassay and application to rubella antibody detection.

Bacterial luciferase has been modified with the thiolating reagent S-acetylmercaptosuccinic anhydride and covalently crosslinked to either Staphylococcus aureus protein A or anti-human immunoglobulin G (IgG) with the heterobifunctional reagent m-maleimidobenzoic acid N-hydroxysuccinimide ester. The conjugates retain enzymatic light-emitting activity and have the ability to bind IgG antibody. The ability of these conjugates to detect human IgG has been demonstrated by application to rubella immunity screening. Rubella antibodies are isolated from serum on the surface of rubella antigen-coated tubes and subsequently determined by light emitted from bound conjugate. In a preliminary study, the bioluminescent immunoassay has been compared to a commercial rubella antibody radioimmunoassay and found to be comparable in the ability to determine rubella immunity.

The utilization of protein from a low-protein diet as determined by limiting essential amino acids.

Protein utilization was determined by assay with low-protein diets limiting in one of nine essential amino acids. The diets contained 50 g protein/kg and the amino acid score of the protein was 0.50. Protein utilization was found to be related to the particular limiting essential amino acids present. The usefulness of Bender's (1965) standard amino acid requirement for the estimation of amino acid utilization from a low-protein diet is discussed.

Sugar transport by the bacterial phosphotransferase system. Preparation of a fluorescein derivative of the glucose-specific phosphocarrier protein IIIGlc and its binding to the phosphocarrier protein HPr.

In diauxic growth, the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) regulates the utilization of certain compounds which are not PTS substrates. It has recently been shown that this PTS regulation is mediated via one of the PTS phosphocarrier proteins, IIIGlc. In the present studies, IIIGlc was derivatized with the fluorescent reagent fluorescein-5-isothiocyanate. One mol of label was incorporated per mol of protein and the label was located at the NH2-terminal amine, as shown by tryptic peptide mapping and one-step Edman-type degradation. The fluorescent moiety was found to be stable and resistant to photodecomposition. The fluorescent IIIGlc was purified and shown to be fully active in its ability to accept phosphate from phospho-HPr (the histidine-containing phosphocarrier protein of the phosphotransferase system), but only 20% active in catalyzing the transfer of the phosphate to methyl alpha-glucoside via the membrane-bound II-BGlc protein. The decay of the fluorescence intensity was dominated by a single component (90%) with a lifetime of 4 ns. The decay of the fluorescence emission anisotropy was determined for excitation in both a negative and positive transition of fluorescein and was best described in terms of a biexponential function, indicating internal motion of the fluorophore and possible anisotropic rotation of the protein as a whole. The formation of a complex between IIIGlc and HPr was demonstrated by using the techniques of time-resolved and steady state fluorescence emission measurements, resonance energy transfer, and equilibrium gel filtration.

Properties and uses of immobilized light-emitting enzyme systems from Beneckea harveyi.

Bacterial luciferase and NADH:FMN oxidoreductase have been immobilized onto arylamine glass beads. These immobilized enzymes can detect as little as 0.2 pmol of NADH per assay sample. Glucose-6-phosphate dehydrogenase has been co-immobilized with these enzymes, and with this system it is possible to quantitate 1 pmol of glucose 6-phosphate. By co-immobilizing a fourth enzyme, hexokinase, onto the glass beads, the system can reproducibly detect 20 pmol of glucose per liter. These immobilized enzyme systems are potentially superior to soluble enzymes by being reusable and much more stable. We compared the light-emitting properties of the immobilized enzyme systems with that of an equivalent mixture of the soluble enzymes. The most striking difference was the apparently more efficient conversion of NADH or glucose 6-phosphate to light by the immobilized enzymes. We used hydroxysteroid dehydrogenase in developing a soluble coupled system for the assay of androsterone and testosterone. The lower limit of detection was 100 pmol.

Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.

Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.