Stein and Moore Award address. The molten globule intermediate of apomyoglobin and the process of protein folding.

The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of alpha-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N<-->I<-->U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagnesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., & Baldwin, R.L., 1991, Biochemistry 30, 4113-4118), which can explain why I is formed rapidly from U at the start of folding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caffeine Junkie: an Unprecedented Glutathione S-Transferase-Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5

Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N₁- and N₃-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N₇-demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) and Nuclear Factor-κB (NF-κB): A FEED-FORWARD LOOP FOR SYSTEMIC AND VASCULAR INFLAMMATION*

The interaction between vascular cells and macrophages is critical during vascular remodeling. Here we report that the scaffolding protein, ezrin-binding phosphoprotein 50 (EBP50), is a central regulator of macrophage and vascular smooth muscle cells (VSMC) function. EBP50 is up-regulated in intimal VSMC following endoluminal injury and promotes neointima formation. However, the mechanisms underlying these effects are not fully understood. Because of the fundamental role that inflammation plays in vascular diseases, we hypothesized that EBP50 mediates macrophage activation and the response of vessels to inflammation. Indeed, EBP50 expression increased in primary macrophages and VSMC, and in the aorta of mice, upon treatment with LPS or TNFα. This increase was nuclear factor-κB (NF-κB)-dependent. Conversely, activation of NF-κB was impaired in EBP50-null VSMC and macrophages. We found that inflammatory stimuli promote the formation of an EBP50-PKCζ complex at the cell membrane that induces NF-κB signaling. Macrophage activation and vascular inflammation after acute LPS treatment were reduced in EBP50-null cells and mice as compared with WT. Furthermore, macrophage recruitment to vascular lesions was significantly reduced in EBP50 knock-out mice. Thus, EBP50 and NF-κB participate in a feed-forward loop leading to increased macrophage activation and enhanced response of vascular cells to inflammation.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Quirks of dye nomenclature. 6. Malachite green

Malachite green was discovered independently by two researchers in Germany in the 19^th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed.     

Escalation and extinction selectivity: morphology versus isotopic reconstruction of bivalve metabolism

Studies that have tested and failed to support the hypothesis that escalated species (e.g., those with predation-resistant adaptations) are more susceptible to elimination during mass extinctions have concentrated on the distribution and degree of morphological defenses in molluscan species. This morphological approach to determining level of escalation in bivalves may be oversimplified because it does not account for metabolic rate, which is an important measure of escalation that is less readily accessible for fossils. Shell growth rates in living bivalves are positively correlated with metabolic rate and thus are potential indicators of level of escalation. To evaluate this approach, we used oxygen isotopes to reconstruct shell growth rates for two bivalve species (Macrocallista marylandica and Glossus markoei) from Miocene-aged sediments of Maryland. Although both species are classified as non-escalated based on morphology, the isotopic data indicate that M. marylandica was a faster-growing species with a higher metabolic rate and G. markoei was a slower-growing species with a lower metabolic rate. Based on these results, we predict that some morphologically non-escalated species in previous tests of extinction selectivity should be reclassified as escalated because of their fast shell growth rates (i.e., high metabolic rates). Studies that evaluate the level of escalation of a fauna should take into account the energetic physiology of taxa to avoid misleading results.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.