Individuals homozygous for the age-related macular degeneration risk-conferring variant of complement factor H have elevated levels of CRP in the choroid.

Polymorphisms in the complement factor H gene (CFH) are associated with a significantly increased risk for, or protection against, the development of age-related macular degeneration (AMD). The most documented risk-conferring single-nucleotide polymorphism results in a tyrosine-to-histidine substitution at position 402 (Y402H) of the CFH protein. In this work, we examined the ocular distributions and relative abundance of CFH, several CFH-binding proteins, and abundant serum proteins in the retinal pigmented epithelium (RPE), Bruch's membrane, and choroid (RPE-choroid) in CFH homozygotes possessing either the "at-risk" 402HH or "normal" 402YY variants. Although CFH immunoreactivity is high in the choroid and in drusen, no differences in CFH-labeling patterns between genotypes are apparent. In contrast, at-risk individuals have significantly higher levels of the CFH-binding protein, C-reactive protein (CRP), in the choroidal stroma. Immunoblots confirm that at-risk individuals have approximately 2.5-fold higher levels of CRP in the RPE-choroid; no significant differences in the levels of CFH or other serum proteins are detected. Similarly, we find no differences in CFH transcription levels in the RPE-choroid nor evidence for local ocular CRP transcription. Increased levels of CRP in the choroid may reflect a state of chronic inflammation that is a by-product of attenuated CFH complement-inhibitory activity in those who possess the CFH at-risk allele. Because the CRP-binding site in CFH lies within the domain containing the Y402H polymorphism, it is also possible that the AMD risk-conferring allele alters the binding properties of CFH, thereby leading to choroidal CRP deposition, contributing to AMD pathogenesis.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration.

Age-related macular degeneration (AMD) is a blinding disease that afflicts millions of adults in the Western world. Although it has been proposed that a threshold event occurs during normal aging which leads to AMD, the sequelae of biochemical, cellular, and/or molecular events leading to the development of AMD are poorly understood. Although available data provide strong evidence that a significant proportion of AMD has a genetic basis, no gene(s) has yet been identified that causes a significant proportion of AMD. Moreover, no major molecular pathways involved in the etiology of this disease have been elucidated.Drusen, pathological deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane, are significant risk factors for the development of AMD. In our view, the development of testable new hypotheses of drusen origins has been hindered significantly by the absence of a comprehensive profile of their molecular composition. In this review, we describe an integrated ultrastructural, histochemical, molecular biological, and biochemical approach to identify specific molecular pathways associated with drusen biogenesis. The implicit assumption underlying these recent investigations has been that a thorough understanding of the composition of drusen and source(s) of drusen-associated material is likely to provide fresh insight into the pathobiology underlying AMD. Significantly, these studies have revealed that proteins associated with inflammation and immune-mediated processes are prevalent among drusen-associated constituents. Transcripts that encode a number of these molecules have been detected in retinal, RPE, and choroidal cells. These data have also lead to the observations that dendritic cells, potent antigen-presenting cells, are intimately associated with drusen development and that complement activation is a key pathway that is active both within drusen and along the RPE-choroid interface. We propose herein a unifying hypothesis of drusen biogenesis that attempts to incorporate a large body of new and previously published structural, histochemical, and molecular data pertaining to drusen composition and development. This theory is put forth with the acknowledgment that numerous AMD genotypes may exist. Thus, only some aspects of the proposed hypothesis may be involved in any given AMD genotype. Importantly, this hypothesis invokes, for the first time, the potential for a direct role of cell- and immune-mediated processes in drusen biogenesis. We acknowledge that the proposed hypothesis clearly represents a paradigm shift in our conceptualization pertaining to pathways that participate in the development of drusen and age-related macular degeneration. It is our hope that other investigators will test, validate and/or refute various aspects of this hypothesis, and in so doing, increase our overall understanding of the biological pathways associated with early AMD.

Phosphorylation of human N-myristoyltransferase by N-myristoylated SRC family tyrosine kinase members.

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the amino terminal glycine residue of a number of important proteins especially the non-receptor tyrosine kinases whose activity is important for tumorigenesis. Human NMT was found to be phosphorylated by non-receptor tyrosine kinase family members of Lyn, Fyn and Lck and dephosphorylated by the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin. Deletion of 149 amino acids from the N-terminal end resulted in the absence of phosphorylation suggesting that the phosphorylation sites are located in the N-terminal end of NMT. Furthermore, a site-directed mutagenesis study indicated that substitution of tyrosine 100 with phenylalanine served NMT as a poor substrate for the Lyn kinase. A synthetic peptide corresponding to the amino-terminal region encompassing tyrosine 100 of NMT served as a good substrate for the Lyn and Fyn kinases. Our studies also indicated that NMT was found to interact with Lyn through its N-terminal end in a phosphorylation-dependent manner. This is the first study demonstrating the cross-talk between NMT and their myristoylated protein substrates in signaling pathways.

Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation.

PURPOSE: The inheritance of specific apolipoprotein E allelles has been linked to atherosclerosis, Alzheimer disease, and, most recently, to the incidence of age-related macular degeneration. Apolipoprotein E is a common component of the extracellular plaques and deposits characteristic of these disorders, including drusen, which are a hallmark of age-related macular degeneration. Accordingly, we assessed the potential biosynthetic contribution of local ocular cell types to the apolipoprotein E found in drusen. METHODS: We measured apolipoprotein E mRNA levels in human donor tissues using a quantitative assay of apolipoprotein E transcription, and we localized apolipoprotein E protein to specific cell types and compartments in the neural retina, retinal pigmented epithelium, and choroid using laser scanning confocal immunofluorescence microscopy. RESULTS: Apolipoprotein E immunoreactivity is associated with photoreceptor outer segments, the retinal ganglion cell layer, the retinal pigmented epithelium basal cytoplasm and basal lamina, and with both collagenous layers of Bruch membrane. Apolipoprotein E appears to be a ubiquitous component of drusen, irrespective of clinical phenotype. It also accumulates in the cytoplasm of a subpopulation of retinal pigmented epithelial cells, many of which overlie or flank drusen. Mean levels of apolipoprotein E mRNA in the adult human retina are 45% and 150% of the levels measured in liver and adult brain, the two most abundant biosynthetic sources of apolipoprotein E. Apolipoprotein E mRNA levels are highest in the inner retina, and lowest in the outer retina where photoreceptors predominate. Significant levels of apolipoprotein E mRNA are also present in the retinal pigmented epithelium/choroid complex and in cultured human retinal pigmented epithelial cells. CONCLUSIONS: Apolipoprotein E protein is strategically located at the same anatomic locus where drusen are situated, and the retinal pigmented epithelium is the most likely local biosynthetic source of apolipoprotein E at that location. Age-related alteration of lipoprotein biosynthesis and/or processing at the level of the retinal pigmented epithelium and/or Bruch membrane may be a significant contributing factor in drusen formation and age-related macular degeneration pathogenesis.

An interfacial mechanism and a class of inhibitors inferred from two crystal structures of the Mycobacterium tuberculosis 30 kDa major secretory protein (Antigen 85B), a mycolyl transferase.

The Mycobacterium tuberculosis 30 kDa major secretory protein (antigen 85B) is the most abundant protein exported by M. tuberculosis, as well as a potent immunoprotective antigen and a leading drug target. A mycolyl transferase of 285 residues, it is closely related to two other mycolyl transferases, each of molecular mass 32 kDa: antigen 85A and antigen 85C. All three catalyze transfer of the fatty acid mycolate from one trehalose monomycolate to another, resulting in trehalose dimycolate and free trehalose, thus helping to build the bacterial cell wall. We have determined two crystal structures of M. tuberculosis antigen 85B (ag85B), initially by molecular replacement using antigen 85C as a probe. The apo ag85B model is refined against 1.8 A data, to an R-factor of 0.196 (R(free) is 0.276), and includes all residues except the N-terminal Phe. The active site immobilizes a molecule of the cryoprotectant 2-methyl-2,4-pentanediol. Crystal growth with addition of trehalose resulted in a second ag85B crystal structure (1.9 A resolution; R-factor is 0.195; R(free) is 0.285). Trehalose binds in two sites at opposite ends of the active-site cleft. In our proposed mechanism model, the trehalose at the active site Ser126 represents the trehalose liberated by temporary esterification of Ser126, while the other trehalose represents the incoming trehalose monomycolate just prior to swinging over to the first trehalose site to displace the mycolate from its serine ester. Our proposed interfacial mechanism minimizes aqueous exposure of the apolar mycolates. Based on the trehalose-bound structure, we suggest a new class of antituberculous drugs, made by connecting two trehalose molecules by an amphipathic linker.

Complement activation and inflammatory processes in Drusen formation and age related macular degeneration.

Recent studies implicate inflammation and complement mediated attack as early events in drusen biogenesis. The investigations described here sought to determine whether primary sites of complement activation could be identified within drusen substructure, and whether known inhibitors of the terminal pathway of complement are present in drusen and/or retinal pigmented epithelial (RPE) cells that lie in close proximity to drusen. Immunohistochemical examination shows two fluid phase regulators of the terminal pathway, vitronectin (Vn, S-protein) and clusterin (apolipoprotein J), to be present in drusen; Vn also accumulates in the cytoplasm of RPE cells that are closely associated with drusen. The membrane associated complement inhibitor, complement receptor 1, is also localized in drusen, but it is not detected in RPE cells immunohistochemically. In contrast, a second membrane associated complement inhibitor, membrane cofactor protein, is present in drusen associated RPE cells, as well as in small, spherical substructural elements within drusen. These previously unidentified elements also show strong immunoreactivity for proteolytic fragments of complement component C3 that are characteristically deposited at sites of complement activation. It is proposed that these structures represent residual debris from degenerating RPE cells that are the targets of complement attack. It is likely that RPE cell debris entrapped between the RPE monolayer and Bruch's membrane serves as a chronic inflammatory stimulus and a potential nucleation site for drusen formation. Thus, the process of drusen biogenesis may be envisaged as a secondary manifestation of primary RPE pathology that is exacerbated by consequences of local inflammatory processes.

Using a phage display library to identify basic residues in A-Raf required to mediate binding to the Src homology 2 domains of the p85 subunit of phosphatidylinositol 3'-kinase.

Src homology 2 (SH2) domains are found in a variety of cytoplasmic proteins involved in mediating signals from cell surface receptors to various intracellular pathways. They fold as modular units and are capable of recognizing and binding to short linear peptide sequences containing a phosphorylated tyrosine residue. Here we show that each of the SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase selects phage displayed peptide sequences containing the core (L/I)-A-(R/K)-I-R. The serine/threonine kinase A-Raf, containing the sequence LQRIRS, is associated with the p85 protein in both quiescent and growth factor stimulated cells. This suggests that p85 and A-Raf exist in a protein complex in cells and that complex formation does not require growth factor stimulation. We also show that p85 and A-Raf can bind directly to each other in vitro and that this interaction is mediated in part by the p85 SH2 domains. Further, the p85 SH2 domains require at least one of four distinct basic-X-basic sequence motifs within A-Raf for binding. This is the first description of a phosphotyrosine-independent SH2 domain interaction that requires basic residues on the SH2 ligand.

A potential role for immune complex pathogenesis in drusen formation.

Drusen are abnormal extracellular deposits that accumulate between the retinal pigmented epithelium and Bruch's membrane and are commonly associated with age-related macular degeneration. Our recent work has identified a number of plasma proteins as molecular components of drusen. Of interest is the fact that many of these drusen-associated molecules are acute phase reactant proteins and some have established roles in mediating immune responsiveness. As immune and inflammatory responses appear to play a role in the formation of other pathologic age-related deposits, we examined the distribution of immunoglobulin molecules and terminal complement complexes at sites of drusen deposition. Here, we report that concentrations of immunoglobulin G and terminal C5b-9 complement complexes are present in drusen. In addition, we observe that retinal pigmented epithelial cells overlying or directly adjacent to drusen, as well as some within apparently normal epithelia, exhibit cytoplasmic immunoreactivity for immunoglobulin and the C5 component of complement. Taken together, these results suggest that drusen biogenesis may be a byproduct of immune responsiveness, and they implicate immune complex-mediated pathogenesis involving retinal pigmented epithelial cells as an initiating event in drusen formation.

Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease.

Age-related macular degeneration (AMD), a blinding disorder that compromises central vision, is characterized by the accumulation of extracellular deposits, termed drusen, between the retinal pigmented epithelium and the choroid. Recent studies in this laboratory revealed that vitronectin is a major component of drusen. Because vitronectin is also a constituent of abnormal deposits associated with a variety of diseases, drusen from human donor eyes were examined for compositional similarities with other extracellular disease deposits. Thirty-four antibodies to 29 different proteins or protein complexes were tested for immunoreactivity with hard and soft drusen phenotypes. These analyses provide a partial profile of the molecular composition of drusen. Serum amyloid P component, apolipoprotein E, immunoglobulin light chains, Factor X, and complement proteins (C5 and C5b-9 complex) were identified in all drusen phenotypes. Transcripts encoding some of these molecules were also found to be synthesized by the retina, retinal pigmented epithelium, and/or choroid. The compositional similarity between drusen and other disease deposits may be significant in view of the recently established correlation between AMD and atherosclerosis. This study suggests that similar pathways may be involved in the etiologies of AMD and other age-related diseases.

Rapid upregulation of fibroblast growth factor receptor 1 (flg) by rat photoreceptor cells after injury.

PURPOSE: To determine the mechanism by which basic fibroblast growth factor (bFGF) exerts its neuroprotective effects on degenerating or injured photoreceptors. METHODS: Confocal immunofluorescence microscopy was used to identify sites of bFGF and FGF receptor 1 (FGFR1) expression after focal injury or experimental retinal detachment in adult rats. FGFR1 expression was analyzed immunohistochemically and at the transcription level in single photoreceptor cells, after reverse transcription (RT), using the polymerase chain reaction (PCR). Real time quantitative RT-PCR was used to measure changes in FGFR1 mRNA levels in the retina in response to injury or detachment. RESULTS: Confocal immunofluorescence observations showed that FGFR1 immunoreactivity in the rat retina is concentrated primarily in the perinuclear cytoplasm of photoreceptor cell bodies. Reverse transcription of total RNA derived from dissociated rat photoreceptor cells, followed by amplification of FGFR1 cDNA using the PCR, verified the presence of FGFR1 transcripts in normal rat photoreceptor cells; in contrast, no evidence of bFGF transcription was detected. Collectively, these results provide compelling evidence for FGFR1 gene expression by rat photoreceptors in situ. Within hours after experimental retinal detachment or focal injury, there is a twofold increase in FGFR1 immunoreactivity in the outer nuclear layer that persists for at least 7 days; a similar increase in bFGF immunoreactivity in the interphotoreceptor matrix is also apparent. This increase in FGFR1 protein levels after detachment and injury also was confirmed by western blot analysis. Real time quantitative RT-PCR analyses revealed that a rapid upregulation of FGFR1 mRNA occurred within 12 hours after retinal injury/detachment, but then declined to near baseline levels by 24 hours. CONCLUSIONS: This body of evidence strongly suggests that the photoreceptor rescue effect elicited by retinal injury as well as by injection of exogenous bFGF is mediated, at least in part, by upregulation of the FGFR1 by the photoreceptor cells.

Vitronectin gene expression in the adult human retina.

PURPOSE: To determine whether vitronectin (Vn), a plasma protein and extracellular matrix molecule that is also a prominent constituent of drusen, is synthesized by cells in the adult human retina. METHODS: The distribution of Vn in the normal adult human retina was examined using antibodies to circulating plasma Vn and to the multimeric, heparin-binding form that is most prevalent in extravascular tissues. Evidence of Vn transcription by retinal cells was analyzed by in situ hybridization and also by reverse transcription of total RNA derived from dissociated human or mouse photoreceptors followed by amplification using polymerase chain reaction (RT-PCR). RESULTS: Cytoplasmic immunoreactivity for plasma Vn or multimeric Vn was detected in photoreceptors, in a subpopulation of neurons situated in the inner retina, and in vitreous hyalocytes. Extracellular labeling was limited primarily to Bruch's membrane and the retinal vasculature. At the transcriptional level, Vn mRNA was localized to both photoreceptors and ganglion cells by in situ hybridization. The in situ findings were corroborated by RT-PCR using total RNA from dissociated mouse or human photoreceptor cells. CONCLUSIONS: The results constitute the first evidence for Vn gene expression by adult neurons in the mammalian central nervous system. The identification of the photoreceptors as a cellular source of Vn suggests that these cells have the potential to make a biosynthetic contribution to the Vn that is found in drusen.

Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).

Centrosymmetric bilayers in the 0.75 A resolution structure of a designed alpha-helical peptide, D,L-Alpha-1.

We report the 0.75 A crystal structure of a racemic mixture of the 12-residue designed peptide "Alpha-1" (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1bar. The unit cell contains two L-alpha-helices and two D-alpha-helices. The columnar-sheet bilayer motif seen in L-Alpha-1 is maintained in the D,L-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.

The human retina and retinal pigment epithelium are abundant sources of vitronectin mRNA.

Vitronectin (Vn), a multifunctional plasma protein synthesized primarily in the liver, is often present as a component of the extracellular plaques and deposits that accompany various age-related human diseases. Recently, we reported that Vn is also a prominent molecular constituent of drusen, the extracellular deposits associated with age-related macular degeneration (AMD) (1). The cellular source(s) of the Vn in drusen, as well as in these other plaques and deposits, remains uncertain. In this study, we used real-time quantitative RT-PCR to measure the relative levels of Vn mRNA in the cells and tissues that lie in close proximity to drusen. The results confirm that the human liver is an abundant source of Vn mRNA. Levels of Vn mRNA in kidney, lung, and fetal or adult brain are <3% of those in liver. Remarkably, mean Vn mRNA levels in the neural retina significantly exceed those in brain and represent close to 40% of the Vn mRNA value measured in human liver. Substantial levels of Vn mRNA are also present in the adjacent retinal pigment epithelium (RPE). These results identify the neural retina, for the first time, as an abundant source of Vn mRNA. They also suggest that both the neural retina and RPE are potent biosynthetic sources of Vn in humans, and potentially significant local contributors to the Vn that accumulates in drusen.

Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.

Age-related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruch's membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen-containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT-PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)-choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD.

v-fps causes transformation by inducing tyrosine phosphorylation and activation of the PDGFbeta receptor.

The v-fps oncogene encodes an activated tyrosine kinase which is capable of transforming fibroblasts. In this report, we provide evidence that within a few minutes of activation of the tyrosine kinase activity of v-Fps, tyrosine phosphorylation of the platelet derived growth factor (PDGF) beta receptor is observed. Further, sustained expression of activated v-Fps results in a down-regulation of the PDGF receptor both at the level of the mRNA (approximately 4-8-fold), but even more markedly at the level of the receptor protein (> 100-fold). The kinase activity of the v-Fps oncoprotein was found to be required for both the induction of PDGF receptor tyrosine phosphorylation and ultimately the reduced receptor protein levels. Tyrosine phosphorylation of a kinase inactive PDGF receptor was also demonstrated in cells which also express v-fps, but this was not sufficient to induce transformation. Only cells expressing both v-fps and a wild type PDGF receptor were able to form colonies in soft agar. These findings suggest that wild type v-fps may use tyrosine phosphorylation of the PDGFbeta receptor to constitutively activate the kinase activity of the receptor, resulting in a sustained proliferative signal and fibroblast transformation.

Conditioned flavour aversions in sheep: the relationship between the dose rate of a secondary plant compound and the acquisition and persistence of aversions.

Within the overall objective of whether ruminants are able to form conditioned aversions (CFA) toward a food flavour associated with the administration of an aversive stimulus which occurs naturally in food plants (oxalic acid, OA), two specific objectives were tested: (1) whether the rate and degree of formation of CFA are dependent on the dose rate of OA administered and (2) whether the persistence of formed CFA depends on the previous dose rate of OA. Sheep were conditioned to associate the specific flavour of one of two novel foods with either the oral administration of OA or equivalent placebos. Four dose rates of OA were tested (0.06, 0.12, 0.18 and 0.24 g/kg sheep live weight per d), with twelve sheep per dose. Each conditioning period lasted for 8 d and was repeated four times. At the end of each conditioning period the preference for the two flavours was measured in short-term, 20 min preference tests. The persistence of the CFA was measured at 0, 7, 21 and 49 d after the completion of the conditioning phase with long-term, 3 h preference tests. The results of the experiment indicated that: (1) the rate and degree of formation of CFA were dependent on the rate of administration of OA; (2) sheep required repeated exposures to the lower dose rates of OA in order to develop CFA and these CFA did not persist in the absence of continual reinforcement; (3) CFA to the higher dose rates of OA were developed after as little as one exposure and persisted over a period of at least 7 weeks. These findings are consistent with the expectation that ruminants should be able to select a diet which minimizes the risk of consumption of potentially harmful foods, whilst at the same time maintaining a degree of flexibility in their feeding behaviour.

Charges, hydrogen bonds, and correlated motions in the 1 A resolution refined structure of the mating pheromone Er-1 from Euplotes raikovi.

A detailed description is given of the structure of the small protein mating pheromone Er-1 at atomic resolution. Emphasis is placed on the locations of charges and hydrogen bonds. The model includes all the protein atoms, anisotropic displacement parameters, four disordered side chains, 22 water molecules, a disordered ethanol, and "riding" hydrogen atoms. Analysis of the model revealed that this dense crystal is perfused by hydrogen-bonding networks of solvent and protein atoms. The termini of helices are capped by hydrogen bonding to solvent and protein atoms, and to symmetry-related molecules. An examination of the valencies and charges of the hydrogen-bonding groups suggests that three of the "water" molecules capping the C termini of two helices, and one other, may instead be NH4 ions. Water molecules mediate all but one of the interhelical hydrogen bonds, and many of the lattice interactions. Regions of the molecule where the atomic vibrations deviate from isotropy are identified. There is almost no overall libration of the molecule allowed by the packing, but the side-chains vibrate relative to the backbone. Four side-chains display alternate conformations. Indirect evidence is presented that the switches between their conformations are correlated and driven by protonation of acidic side-chains. These structural features are discussed in the context of function and stability. Equipped with the analysis of the model, we review the course and results of the refinement of the model against 1 A X-ray diffraction data to a crystallographic R-factor of 12.92%.

Characterization of drusen-associated glycoconjugates.

BACKGROUND: Drusen are extracellular deposits that accumulate between the basal lamina of the retinal pigment epithelium and the elastic lamina of Bruch membrane in aging human eyes. Although specific types of drusen are recognized as significant risk factors for the development of both the atrophic and exudative forms of age-related macular degeneration, few studies have focused on defining their molecular composition. As an initial step toward identifying the molecular composition of drusen, assessing the biochemical relation between hard and soft drusen, and identifying potential target molecules for detecting drusen clinically, the authors have analyzed their carbohydrate composition using lectin histochemistry. METHODS: Sections of eyes from human donors containing a spectrum of hard and soft drusen were screened with a battery of 22 fluorescein-conjugated lectins. RESULTS: A specific subset of six lectins bind drusen intensely. No significant differences in lectin binding are observed between any subclass of hard and soft drusen. Some drusen exhibit homogeneous, uniform labeling, whereas others show asymmetrical, heterogeneous distribution of glycoconjugates. CONCLUSION: This study shows that glycoconjugates containing specific carbohydrate residues are present in all classes of hard and soft drusen examined. The observation that hard and soft drusen are bound by the same lectins suggests that they may be related compositionally. Identification of the drusen-associated glycoconjugates shown in this investigation will facilitate studies of drusen genesis and their involvement in the pathogenesis of age-related maculopathy. They may also provide a basis for developing avenues of therapeutic intervention.