Molecular features of the copper binding sites in the octarepeat domain of the prion protein

Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH approximately 6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.     

Preparation and investigation of antibacterial carbohydrate-based surfaces

Surfaces bearing carbohydrate units have been modified in a two-step process to incorporate functionalities (lipophilic with polycationic units) that bear antibacterial activity. The effectiveness of these modified surfaces for antibacterial action against a series of seven Gram-positive and Gram-negative bacteria are reported.     

Isolation and characterization of a stable 2,4-dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus, using chemostat culture

A strain of Variovorax paradoxus degrading 2,4-dichlorophenoxyacetic acid (2,4-D) was isolated from the Dijon area (France) using continuous chemostat culture. This strain, designated TV1, grew on up to 5 mM 2,4-D and efficiently degraded the herbicide as sole carbon source as well as in presence of soil extracts. It also degraded phenol and 2-methyl, 4-chlorophenoxyacetic acid at 3 mM and 2,4-dichlorophenol at 1 mM. This organism contained a stable 200 kb plasmid, designated pTV1, which showed no similarity in its restriction pattern with the archetypal 2,4-D catabolic plasmid pJP4. However, pTV1 contained an 11 kb BamHI fragment which hybridized at low stringency with the 2,4-D degradative genes tfdA, tfdB and tfdR from pJP4. PTV1 partial tfdA sequence showed 77 % similarity with the archetypal tfdA gene sequence from Ralstonia eutropha JMP134. Tn5 mutagenesis confirmed the involvement of this gene in the 2,4-D catabolic pathway. © Rapid Science Ltd. 1998     

The effects of dietary boric acid on bone strength in rats

The effects of dietary boron (B) (from boric acid [BA]) on bone strength were evaluated using male F344 rats. B was administered by dietary admixture of BA to NIH-07 feed at concentrations of 200, 1000, 3000, and 9000 ppm. The latter two levels were found in previous studies to be reproductively toxic to both males and the developing fetus. The first two levels are below and just at, respectively, the levels for producing fetal malformations, and are below the dose required to produce male reproductive toxicity. Resistance to destructive testing was measured on femora, tibiae, and lumbar vertebrae. Although femur and tibia resistance to bending force were not affected by any amount of dietary B, vertebral resistance to a crushing force was increased by ≈10%, at all dose levels (200-9000 ppm). These data show that even levels of BA that are not reproductively toxic can affect the strength of the axial skeleton in rats.     

Injury-Related Factors and Conditions Down-Regulate the Thrombin Receptor (PAR-1) in a Human Neuronal Cell Line

Abstract: Previous studies have demonstrated that thrombin can induce potent effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR-1). Mechanisms of PAR-1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR-1 regulation in neural cells. In the present study, PAR-1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti-PAR-1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high-affinity, specific binding to a single affinity class of receptors: K_D = 3.13 and 5.25 nM, B_max = 190.1 and 67.8 fmol/mg of protein for ¹²⁵I-ATAP2 and ¹²⁵I-SPAN12, respectively. Specificity for PAR-1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR-1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin-induced PAR-1 cleavage and internalization, with protein synthesis-dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR-1 expression. Significant decreases in PAR-1 expression were induced by the protein kinase C activator phorbol 12-myristate 13-acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (32% at 3 h), and the injury-related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally with PAR-1 cell surface expression.     

Phenotypic and Phylogenetic Characterization of Ruminal Tannin-Tolerant Bacteria

The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (Ovis aries), Honduran and Colombian goats (Capra hircus), white-tail deer (Odocoileus virginianus) from upstate New York, and Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus Streptococcus and were most closely related to ruminal strains of Streptococcus bovis and the recently described organism Streptococcus gallolyticus. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family Enterobacteriaceae in the gamma subdivision of the class Proteobacteria. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed.The toxicity of phenolic compounds in the environment has fostered studies of bacteria that are able to tolerate and/or metabolize high levels of these compounds, particularly under anaerobic conditions (1, 4, 14, 21, 30, 36). Tannins are secondary polyphenolic compounds known primarily for their ability to bind to and precipitate proteins and other macromolecules. Tannins have been found in many habitats, including sewage sludge, forest litter, and the rumen (9, 14, 15, 28). Bacteria capable of degrading or tolerating tannins have been isolated from sewage sludge (14) and from the alimentary tracts of koalas (Phascolarctos cinereus) (33), goats (Capra hircus) (4, 30), and horses (Equus caballus) (31). Most of the isolates have been characterized phenotypically, and phylogenetic characterization has been limited to studies conducted in Australia (4, 34, 35) and Japan (31). Little is known about the geographic diversity and host species diversity of tannin-tolerant and tannin-degrading bacteria.The objective of this study was to characterize six recently isolated tannin-tolerant bacteria by examining their phenotypic characteristics and molecular phylogeny. These bacteria were isolated from the ruminal contents of goats (C. hircus), sheep (Ovis aries), white-tail deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni), all of which had consumed forage containing tannins. Our goal was to genetically and biochemically characterize tannin-tolerant bacteria isolated from different host animals in various geographic locations.     

A SecY Homologue Is Required for the Elaboration of the Chloroplast Thylakoid Membrane and for Normal Chloroplast Gene Expression

Results of in vitro and genetic studies have provided evidence for four pathways by which proteins are targeted to the chloroplast thylakoid membrane. Although these pathways are initially engaged by distinct substrates and involve some distinct components, an unresolved issue has been whether multiple pathways converge on a common translocation pore in the membrane. A homologue of eubacterial SecY called cpSecY is localized to the thylakoid membrane. Since SecY is a component of a protein-translocating pore in bacteria, cpSecY likely plays an analogous role. To explore the role of cpSecY, we obtained maize mutants with transposon insertions in the corresponding gene. Null cpSecY mutants exhibit a severe loss of thylakoid membrane, differing in this regard from mutants lacking cpSecA. Therefore, cpSecY function is not limited to a translocation step downstream of cpSecA. The phenotype of cpSecY mutants is also much more pleiotropic than that of double mutants in which both the cpSecA- and ΔpH-dependent thylakoid-targeting pathways are disrupted. Therefore, cpSecY function is likely to extend beyond any role it might play in these targeting pathways. CpSecY mutants also exhibit a defect in chloroplast translation, revealing a link between chloroplast membrane biogenesis and chloroplast gene expression.     

Preparations and properties of tripodal and linear tetradentate N,S-Donor ligands and their complexes containing the MoO22+core

New linear and tripodal tetradentate ligands, LH₂, are reported and their syntheses are described. The new linear ligands L = HSCH₂CH₂SCH₂CH₂NRCH₂CR₂SH, R = H, CH₃) and the new tripodal ligands N(CH₂CH₂SH)₂CH₂Z, Z = CH₂NH₂, CH₂N(CH₃)₂, CH₂N(C₂H₅)₂, CH₂SCH₃ and CO₂^- were synthesized. The known linear ligands HSCH₂CH₂NCH₃(CH₂)_nNCH₃CH₂CH₂SH (n = 2, 3) and HSCR₂CH₂NHCH₂CH₂NHCH₂CR₂SH (R = H, CH₃) were also utilized. These ligands react with MoO₂(acac)₂ in CH₃OH to yield MoO₂L complexes in high yield. Infra-red and ¹H nmr spectra provide evidence to supplement X-ray crystallographic results reported elsewhere for selected numbers of the series. Octahedral structures with cis MoO₂²⁺ groupings are assigned. Solution ¹H nmr studies are consistent with a trans placement of the two thiolate donors in agreement with the X-ray studies.     

An arginyl in the N-terminus of the V1a vasopressin receptor is part of the conformational switch controlling activation by agonist.

Defining how the agonist-receptor interaction differs from that of the antagonist-receptor and understanding the mechanisms of receptor activation are fundamental issues in cell signalling. The V1a vasopressin receptor (V1aR) is a member of a family of related G-protein coupled receptors that are activated by neurohypophysial peptide hormones, including vasopressin (AVP). It has recently been reported that an arginyl in the distal N-terminus of the V1aR is critical for binding agonists but not antagonists. To determine specific features required at this locus to support high affinity agonist binding and second messenger generation, Arg46 was substituted by all other 19 encoded amino acids. Our data establish that there is an absolute requirement for arginyl, as none of the [R46X]V1aR mutant constructs supported high affinity agonist binding and all 19 had defective signalling. In contrast, all of the mutant receptors possessed wildtype binding for both peptide and nonpeptide antagonists. The ratio of Ki to EC50, an indicator of efficacy, was increased for all substitutions. Consequently, although [R46X]V1aR constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype V1aR to become activated. Therefore, in the wildtype V1aR, Arg46 constrains the inactive conformation of the receptor. On binding AVP this constraint is alleviated, promoting the transition to active V1aR. Our findings explain why arginyl is conserved at this locus throughout the evolutionary lineage of the neurohypophysial peptide hormone receptor family of G-protein coupled receptors.     

SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES1

Using flow cytometric analysis of fluorescence, we measured the genome sizes of 18 cultured “free‐living” species and 29 Symbiodinium spp. isolates cultured from stony corals, gorgonians, anemones, jellyfish, and giant clams. Genome size directly correlated with cell size, as documented previously for most eukaryotic cell lines. Among the smallest of dinoflagellates, Symbiodinium spp. (6–15 μm) possessed the lowest DNA content that we measured (1.5–4.8 pg·cell^?1). Bloom‐forming or potentially harmful species in the genera Alexandrium, Karenia, Pfiesteria, and Prorocentrum possessed genomes approximately 2 to 50 times larger in size. A phylogenetic analysis indicated that genome/cell size has apparently increased and decreased repeatedly during the evolution of dinoflagellates. In contrast, genome sizes were relatively consistent across distantly and closely related Symbiodinium spp. This may be the product of intracellular host habitats imposing strong selective pressures that have restricted symbiont size.