Quantitative autoradiographic analysis of [I]-human CGRP binding sites in the dorsal horn of rat following chronic constriction injury or dorsal rhizotomy

Quantitative receptor autoradiography was used to examine the binding of [¹²⁵I]-human CGRP in the dorsal horn of the L4 spinal segment of rats with a chronic constriction injury (CCI) of the sciatic nerve or unilateral dorsal rhizotomies of spinal segments L1–L6. At the times selected for study, we found no change in the amount of CGRP binding in any areas examined following CCI. In contrast, our results showed a temporally related increase in the amount of CGRP binding in areas within laminae I–II and in lateral lamina V of the dorsal horn ipsilateral to the rhizotomies. These results indicate that CGRP binding sites are regulated, most likely, by changes in the release of CGRP. Further, our results suggest that the release of CGRP from primary afferent neurons is unchanged in animals with a CCI.     

Modulation of diphthamide synthesis by 5'-deoxy-5'-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Continuous representations of time-series gene expression data.

We present algorithms for time-series gene expression analysis that permit the principled estimation of unobserved time points, clustering, and dataset alignment. Each expression profile is modeled as a cubic spline (piecewise polynomial) that is estimated from the observed data and every time point influences the overall smooth expression curve. We constrain the spline coefficients of genes in the same class to have similar expression patterns, while also allowing for gene specific parameters. We show that unobserved time points can be reconstructed using our method with 10-15% less error when compared to previous best methods. Our clustering algorithm operates directly on the continuous representations of gene expression profiles, and we demonstrate that this is particularly effective when applied to nonuniformly sampled data. Our continuous alignment algorithm also avoids difficulties encountered by discrete approaches. In particular, our method allows for control of the number of degrees of freedom of the warp through the specification of parameterized functions, which helps to avoid overfitting. We demonstrate that our algorithm produces stable low-error alignments on real expression data and further show a specific application to yeast knock-out data that produces biologically meaningful results.     

Methionine adenosyltransferase activity in cultured cells and in human tissues

We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitt's lymphoma) and EL4 (mouse, T cell lymphoma) cell showed MAT activity near 300 pmol/mg per min. Daudi (human, Burkitt's lymphoma) and almost all monolayer cells had MAT activity below 100 pmol/mg per min. Human peripheral blood lymphocytes had MAT activity of 36 pmol/mg permin. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 pmol/mg per min, while in normal lung it was 2.4 pmol/mg per min.     

Inactivation of liver S-adenosylhomocysteine hydrolase in vitro of rats treated with erythro-9-(2-hydroxynon-3-yl)adenine.

S-Adenosylhomocysteine hydrolase activity decreased in vitro time-dependently in liver homogenates obtained from rats treated in vivo with erythro-9-(2-hydroxynon-3-yl)adenine, a potent inhibitor of adenosine deaminase. The inhibitor in itself had no effect on the stability of the hydrolase. The inactivation of S-adenosylhomocysteine hydrolase was irreversible, proceeded fairly rapidly at a low temperature (0 degrees C) and showed first-order reaction kinetics. Adenosine was found to accumulate in these tissue homogenates during storage. Several lines of evidence suggest that adenosine caused the observed suicide-like inactivation post mortem. Pre-incubation of purified S-adenosylhomocysteine hydrolase at 0 degrees C with adenosine showed a half-maximal inactivation rate at 33 microM substrate concentration; the rate constant of inactivation was 0.01 min-1. Inactivation during tissue preparation and storage complicates the assay of S-adenosylhomocysteine hydrolase activity in samples that contain an inhibitor of adenosine deaminase. These results also suggest that the decrease of S-adenosylhomocysteine hydrolase activity reported to occur in several disturbances of purine metabolism should be re-examined to exclude the possibility of inactivation of the enzyme in vitro.     

Overwintering and migration behaviour of post-spawned Atlantic salmon Salmo salar in a large hydropower-regulated river and reservoir

Tracking 47 post-spawned adult Atlantic salmon Salmo salar L. in a hydropower-regulated river through autumn, winter and spring revealed that winter survival was 56% and 75% in two study years, respectively, with higher mortality of males than females (50% vs. 33% and 100% vs. 13%, respectively). Some kelts (n = 7) displayed nondirected movements that were interpreted as a reconditioning period for an average of 9–17 days prior to directed downstream movements indicating the initiation of migration. Survival after the initiation of migration in spring was 83% and 94% to the hydropower dam in the first and second study years, and decreased to 60 and 63%, respectively, after dam passage. There were no further losses in the downriver reach in the second year, with the first year having a cumulative survival estimate of 53% to the river mouth. Kelts approached the dam when the spillway gates were available as a passage option most of the time (64%–75%), but some kelts arrived at the dam or had not yet passed when spillways were closed (n = 6) and the only remaining passage option was restricted to the turbines. However, all but one kelt that must have passed via turbine were successful in reaching the river mouth. Migratory delay presumably due to searching behaviour caused by low water flow was estimated at approximately 6 days as migration rates were significantly slower in the reservoir (median ± s.e. 8.5 ± 2.5 km day⁻¹) than up- (29.7 ± 5.0 km day⁻¹) or downriver (22.1 ± 3.1 km day⁻¹). The proportion of time (median 30%) that kelts spent swimming upstream (searching behaviour) in the reservoir was a significant variable for migration success.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.     

Food‐web structure and mercury dynamics in a large subarctic lake following multiple species introductions

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Buried charged surface in proteins

BACKGROUND: The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent. Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps. RESULTS: By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed. We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces. In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried. Furthermore, at all sizes few charged groups are fully exposed. As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure. In addition, free energy calculations of stability support the experimental results. CONCLUSIONS: Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment. Consistent with this view, thermophilic proteins often have less buried charge. Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.