Measurement of 1H�C15N and 1H�C13C residual dipolar couplings in nucleic acids from TROSY intensities

Analogous to the recently introduced ARTSY method for measurement of one-bond (1)H-(15)N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base (13)C-(1)H and (15)N-(1)H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in (1)H-(15)N and (13)C-(1)H TROSY-HSQC spectra, and are illustrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for (15)N-(1)H and 65/(S/N) Hz for (13)C-(1)H. The signal-to-noise ratio of both (1)H-(15)N and (1)H-(13)C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with (1)H-(1)H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6° relative to the X-ray structure of the native riboswitch.     

The Caenorhabditis elegans A��1�C42 Model of Alzheimer Disease Predominantly Expresses A��3�C42

Transgenic expression of human amyloid β (Aβ) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, Aβ undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated Aβ is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of Aβ peptide in established transgenic C. elegans lines. Surprisingly, the Aβ being expressed is not full-length 1–42 (amino acids) as expected but rather a 3–42 truncation product. In vitro analysis demonstrates that Aβ_3–42 self-aggregates like Aβ_1–42, but more rapidly, and forms fibrillar structures. Similarly, Aβ_3–42 is also the more potent initiator of Aβ_1–40 aggregation. Seeded aggregation via Aβ_3–42 is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of Aβ expression can now be co-opted to study the proteotoxic effects and processing of Aβ_3–42.Numerous studies support a role for aggregating Aβ³ in mediating the toxicity that underlies AD (1, 2). However, several key questions remain central to understanding how AD and Aβ pathology are related. What is the connection between Aβ aggregation and toxicity? Is there a specific toxic Aβ conformation or species? How and why does aging impact on Aβ precipitation? Significant effort to address these questions has been invested in the use of vertebrate and simple invertebrate model organisms to simulate neurodegenerative diseases through transgenic expression of human Aβ (3). From these models, several novel insights into the proteotoxicity of Aβ have been gained (4–7).Human Aβ (e.g. in brain, cerebrospinal fluid, or plasma) is not found as a single species but rather as diverse mixtures of various modified, truncated, and cross-linked forms (8–10). Specific truncations, covalent modifications, and cross-linked oligomers of Aβ have potentially important roles in determining Aβ-associated neurotoxicity. For example, N-terminal truncations of Aβ have increased abundance in AD, rapidly aggregate, and are neurotoxic (9, 11). Furthermore, the N-terminal glutamic acid residue of Aβ_3–42 can be cyclized to pyroglutamate (Aβ_3(pE)-42) (12), which may be particularly important in AD pathogenesis (13, 14). Aβ_3(pE)-42 is a significant fraction of total Aβ in AD brain (15), accounting for more than 50% of Aβ accumulated in plaques (16). Aβ_3(pE)-42 seeds Aβ aggregation (17), confers proteolytic resistance, and is neurotoxic (13). Recently, glutaminyl cyclase (QC) has been proposed to catalyze, in vivo, pyroglutamate formation of Aβ_3(pE)-40/42 (14, 18). Aβ_1–42 itself cannot be cyclized by QC to Aβ_3(pE)-42 (19), unlike Aβ that commences with an N-terminal glutamic acid-residue (e.g. Aβ_3–42 and Aβ_11–42) (20). QC has broad expression in mammalian brain (21, 22), and its inhibition attenuates accumulation of Aβ_3(pE)-42 into plaques and improves cognition in a transgenic mouse model of AD that overexpresses human amyloid precursor protein (14). N-terminal truncations at position 3 have been reported in senile plaques (23, 24); however, the process that generates Aβ_3–42 is unknown. Currently there are no reported animal models of Aβ_3–42 expression.Advances in surface-enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) analysis now facilitate accurate identification of particular Aβ species. Using this technology, we examined well characterized C. elegans transgenic models of AD that develop amyloid aggregates (25, 26) to see whether the human Aβ they express is post-translationally modified.     

Simultaneous measurement of 1H�C15N and Methyl 1Hm�C13Cm residual dipolar couplings in large proteins

A two-dimensional TROSY-based SIM-(13)C(m)-(1)H(m)/(1)H-(15)N NMR experiment for simultaneous measurements of methyl (1) D (CH) and backbone amide (1) D (NH) residual dipolar couplings (RDC) in {U-[(15)N,(2)H]; Ileδ1-[(13)CH(3)]; Leu,Val-[(13)CH(3)/(12)CD(3)]}-labeled samples of large proteins is described. Significant variation in the alignment tensor of the 82-kDa enzyme Malate synthase G is observed as a function of only slight changes in experimental conditions. The SIM-(13)C(m)-(1)H(m)/(1)H-(15)N data sets provide convenient means of establishing the alignment tensor characteristics via the measurement of (1) D (NH) RDCs in the same protein sample.     

Protease-Activated Receptor�C1 Expression in Rat Microglia after Trimethyltin Treatment

In the nervous system, protease-activated receptors (PARs), which are activated by thrombin and other extracellular proteases, are expressed widely at both neuronal and glial levels and have been shown to be involved in several brain pathologies. As far as the glial receptors are concerned, previous experiments performed in rat hippocampus showed that expression of PAR-1, the prototypic member of the PAR family, increased in astrocytes both in vivo and in vitro following treatment with trimethyltin (TMT). TMT is an organotin compound that induces severe hippocampal neurodegeneration associated with astrocyte and microglia activation. In the present experiments, the authors extended their investigation to microglial cells. In particular, by 7 days following TMT intoxication in vivo, confocal immunofluorescence revealed an evident PAR-1-related specific immunoreactivity in OX-42-positive microglial cells of the CA3 and hilus hippocampal regions. In line with the in vivo results, when primary rat microglial cells were treated in vitro with TMT, a strong upregulation of PAR-1 was observed by immunocytochemistry and Western blot analysis. These data provide further evidence that PAR-1 may be involved in microglial response to brain damage.     

Environment�CKHV�Ccarp�Chuman linkage as a model for environmental diseases

To predict outbreaks of infectious disease and to prevent epidemics, it is essential not only to conduct pathological studies but also to understand the interactions between the environment, pathogen, host and humans that cause and spread infectious diseases. Outbreaks of mass mortality in carp caused by Cyprinid herpesvirus 3 (CyHV-3), formerly known as koi herpesvirus (KHV), disease have occurred worldwide since the late 1990s. We proposed an environment?CKHV?Ccarp?Chuman linkage as a conceptual model for ??environmental diseases?? and specify research subjects that might be necessary to construct and shape this linkage.     

Dominant inheritance and intra�Cfamilial variations in the association of Sturge�CWeber and Klippel�CTrenaunay�CWeber syndromes

This case report shows a genealogical study where a woman has limb hypertrophy and her son has an association of Sturge–Weber syndrome with Klippel–Trenaunay–Weber syndrome. The Sturge–Weber and Klippel–Trenaunay–Weber syndromes appear to be different manifestations of the same affliction. Familial aggregation exists and transmission may be almost imperceptible between generations. Identification of minor manifestations may prove to be a valuable contribution to genetic counseling of families and the prevention of new cases.     

Molecular Cloning of Pigeon UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase and UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Gal��1�C4Gal��1�C4Gal��1�C4GlcNAc Sequence

We previously found that pigeon IgG possesses unique N-glycan structures that contain the Galα1–4Galβ1–4Galβ1–4GlcNAc sequence at their nonreducing termini. This sequence is most likely produced by putative α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1–4Gal and Galβ1–4Gal sequences on the N-glycans, respectively. Because no such glycan structures have been found in mammalian glycoproteins, the biosynthetic enzymes that produce these glycans are likely to have distinct substrate specificities from the known mammalian GalTs. To study these enzymes, we cloned the pigeon liver cDNAs encoding α4GalT and β4GalT by expression cloning and characterized these enzymes using the recombinant proteins. The deduced amino acid sequence of pigeon α4GalT has 58.2% identity to human α4GalT and 68.0 and 66.6% identity to putative α4GalTs from chicken and zebra finch, respectively. Unlike human and putative chicken α4GalTs, which possess globotriosylceramide synthase activity, pigeon α4GalT preferred to catalyze formation of the Galα1–4Gal sequence on glycoproteins. In contrast, the sequence of pigeon β4GalT revealed a type II transmembrane protein consisting of 438 amino acid residues, with no significant homology to the glycosyltransferases so far identified from mammals and chicken. However, hypothetical proteins from zebra finch (78.8% identity), frogs (58.9–60.4%), zebrafish (37.1–43.0%), and spotted green pufferfish (43.3%) were similar to pigeon β4GalT, suggesting that the pigeon β4GalT gene was inherited from the common ancestors of these vertebrates. The sequence analysis revealed that pigeon β4GalT and its homologs form a new family of glycosyltransferases.     

Organic solutes in the deepest phylogenetic branches of the Bacteria: identification of ��(1�C6)glucosyl-��(1�C2)glucosylglycerate in Persephonella marina

The accumulation of organic solutes was investigated in the thermophilic bacteria Persephonella marina and Marinitoga piezophila, two representatives of the deepest lineages in the domain Bacteria. These organisms grow optimally at around 70 °C in medium containing 3 % NaCl. A new disaccharide, accumulating in Persephonella marina, was identified as ??(1?C6)glucosyl-??(1?C2)glucosylglycerate (GGG), by nuclear magnetic resonance. This identification was validated by comparison with the spectra of the compound obtained by chemical synthesis. Besides GGG, the solute pool of Persephonella marina comprised ??-glutamate, di-myo-inositol-1,3??-phosphate and 2-O-??-glucosylglycerate. In contrast, amino acids such as ??-glutamate, proline and alanine were the dominant components of the solute pool of Marinitoga piezophila and sugar derivatives were absent. The ability of GGG to protect protein structure against heat denaturation was assessed using model proteins. A genomic search for the biosynthetic pathways of known ionic solutes in Aquificales and Thermotogales shows the inability of this analysis to predict the nature of compatible solutes and underlines the need for efficient cultivation techniques.