Conformation of the diphtheria toxin T domain in membranes: a site-directed spin-labeling study of the TH8 helix and TL5 loop.

The isolated T domain of diphtheria toxin was mutated by cysteine-scanning mutagenesis at 28 consecutive sites (residues 328-355) that comprise the TH8 helix and the TL5 interhelical loop in the native toxin. After derivatizing the mutant proteins with a sulfhydryl-selective nitroxide reagent, we examined the mobility of each nitroxide and its accessibility to polar and nonpolar paramagnetic reagents, before and after insertion into phospholipid bilayers. The data obtained with the proteins in solution at pH 8 are generally consistent with predictions from the crystal structure of the toxin. Upon membrane binding at pH 4.6, a major structural reorganization of the domain was seen, which dramatically reduced the accessibility of most residues in this region to the polar reagent nickel(II)-ethylenediaminediacetate complex (NiEDDA). Many of these residues also showed reduced accessibility to the nonpolar reagent O(2). Periodic accessibility of the nitroxide side chains along the sequence to these reagents shows that TH8 remains largely helical in the membrane-bound state, with one surface associated with protein and the other facing the hydrophobic interior of the bilayer. In addition, the TL5 loop also appears to become alpha-helical in the membrane, with one surface in contact with protein and the other in contact with the bilayer interior. These findings provide a structural framework for understanding how the T domain forms a transmembrane channel and mediates translocation of diphtheria toxin's enzymic moiety across a membrane.     

Entropy in evolution

Daniel R. Brooks and E. O. Wiley have proposed a theory of evolution in which fitness is merely a rate determining factor. Evolution is driven by non-equilibrium processes which increase the entropy and information content of species together. Evolution can occur without environmental selection, since increased complexity and organization result from the likely capture at the species level of random variations produced at the chemical level. Speciation can occur as the result of variation within the species which decreases the probability of sharing genetic information. Critics of the Brooks-Wiley theory argue that they have abused terminology from information theory and t thermodynamics. In this paper I review the essentials of the theory, and give an account of hierarchical physical information systems within which the theory can be interpreted. I then show how the major conceptual objections can be answered.     

Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding. A site-directed spin labeling study

The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of delta-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an alpha-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes.     

Plant–Environment Interactions in the Low Arctic Torngat Mountains of Labrador

Ecosystems - The eastern Canadian Subarctic and Arctic are experiencing significant environmental change with widespread implications for the people, plants, and animals living there. In this...     

Diphtheria toxin. Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity

Photoaffinity labeling experiments with diphtheria toxin fragment A have implicated glutamic acid 148 as a constituent of the NAD binding site. To evaluate the role of this residue in ADP-ribosylation of elongation factor 2, we replaced it with aspartic acid by in vitro mutagenesis of a toxin gene fragment cloned in Escherichia coli. Fragment A containing aspartic acid at position 148 had less than 0.6% the ADP-ribosylation activity of wild-type fragment A. The mutation produced no change in sensitivity of fragment A to trypsin and little, if any, reduction in affinity of fragment A for NAD. These results indicate that glutamic acid 148 is essential for the ADP-ribosylation of elongation factor 2 and are consistent with other data suggesting that this residue may be at or near the catalytic center of the toxin.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

The extent and significance of petroleum hydrocarbon contamination in Crater Lake,Oregon

In order to evaluate hydrocarbon inputs to Crater Lake from anthropogenic and natural sources, samples of water, aerosol, surface slick and sediment were collected and analyzed by gas chromatography-mass spectrometry (GC-MS) for determination of their aliphatic and aromatic hydrocarbon concentrations and compositions. Results show that hydrocarbons originate from both natural (terrestrial plant waxes and algae) and anthropogenic (petroleum use) sources and are entering the lake through direct input and atmospheric transport. The concentrations of petroleum hydrocarbons range from low to undetectable. The distributions and abundances of n-alkanes, polycyclic aromatic hydrocarbons (PAH) and unresolved complex mixture (UCM) from petroleum are similar for all surface slick sampling sites. The estimated levels of PAH in surface slicks range from 7–9 ng/m² which are low. Transport of petroleum-derived hydrocarbons from the lake surface has resulted in their presence in some sediments, particularly near the boat operations mooring (total petroleum HC = 1440 μg/kg, dry wt. compared to naturally derived n-alkanes, 240 μg/kg, dry wt.). The presence of biomarkers such as the tricyclic terpanes, hopanes and steranes in shallow sediments further confirms petroleum input from boat traffic. In the deep lake sediments, petroleum hydrocarbon concentrations were very low (16 μg/kg, dry wt.). Very low concentrations of PAH were detected in shallow sediments (17–40 μg/kg at 5 m depth near the boat operations) and deep sediments (3–15 μg/kg at 580 m depth). The individual PAH concentrations in sediments (μg/kg or ppb range) are at least three orders of magnitude less than reported threshold effects levels (mg/kg or ppm range, test amphipod Hyalella azteca). Therefore, no adverse effects are expected to occur in benthic biota exposed to these sediments. Boating activities are leaving a detectable level of petroleum in surface waters and lake sediments but these concentrations are very low.     

Drinking among medical students: a questionnaire survey.

To assess the prevalence of drinking among medical students a questionnaire on smoking, exercise, drinking, and weight was distributed among the students available. A total of 260 replies were received from an estimated available population of 350 students (134 men and 126 women). The mean alcohol consumption obtained by a quantity-frequency measure was 20.5 units/week for male students and 14.6 units/week for female students. Retrospective diary reports showed mean (SE) consumptions of 18 (2) units/week for men (n = 134) and 11 (1) units/week for women (n = 126). Consumption among the men closely matched consumption among men matched for age in the general population. Women, however, drank more than women matched for age. Male and female medical students exceeded the suggested maximum for their sex in equal proportions. Quantity-frequency data showed that 31 (23%) men drank over 35 units/week and 28 (22%) women drank over 21 units/week. Of the 59 students exceeding these limits, 51 responded positively to a standard screening questionnaire for alcohol abuse. Forty students reported that they might have a drinking problem, and 138 reported that alcohol had affected their academic performance at some time; 17 of these were affected frequently. The students suggested sensible maximum consumption figures for health education. Smoking was associated with heavy drinking, especially among the women. These results suggest that some medical students are compromising their future health and their academic performance through excessive drinking.     

Recombinant human interleukin 1 alpha: purification and biological characterization

Interleukin 1 (IL 1) is a polypeptide hormone produced by activated macrophages that affects many different cell types involved in immune and inflammatory responses. The cloning and expression of a murine IL 1 cDNA in Escherichia coli encoding a polypeptide precursor of 270 amino acids has been reported, and expression of the carboxy-terminal 156 amino acids of this precursor in E. coli yields biologically active IL 1. By using the murine IL 1 cDNA as a probe, we have isolated its human homolog from cDNA generated to lipopolysaccharide-stimulated human leukocyte mRNA. Nucleotide sequence analysis of this cDNA predicts a protein of analysis of this cDNA predicts a protein of 271 amino acids (termed IL 1 alpha) which shows congruent to 61% homology to its murine counterpart but only 27% homology to a recently characterized human IL 1 precursor (IL 1 beta). We have expressed the carboxy-terminal 154 amino acids of IL 1 alpha in E. coli, purified this protein to homogeneity, and have compared it with pure recombinant murine IL 1 in several different IL 1 assays based on murine and human cells. Recombinant IL 1 is capable of stimulating T cell and fibroblast proliferation and inducing fibroblast collagenase and prostaglandin production, thus proving that a single molecule has many of the activities previously ascribed to only partially purified IL 1 preparations. Our results indicate that there exists a family of at least two human IL 1 genes (alpha and beta) whose dissimilar protein products have similar biological activities.     

Completion of diapause in field populations of the cabbage root fly (Delia radicum)

The various diapause and post-diapause stages entered by cabbage root fly pupae during the overwintering period are shown schematically. Although diapause induction started in mid-Aug., the early-pupating insects did not develop further but were maintained in diapause by the warm autumn temperatures. Therefore, diapause development was simultaneous in all Wellesbourne pupae, whether of second or third generation origin. Diapause development started only in mid-Oct., when mean soil temperatures fell below 10°. In the field, 90% of the overwintering population of cabbage root fly pupae had completed pleted diapause by 5 March 1980, 17 Feb. 1981 and 18 Feb. 1982. This was equivalent to a duration of 19 weeks from mid-Oct. onwards, during the winters of 1979–80, 1980–81 and 1981–82 respectively. A further break between the completion of diapause and the warm conditions required to start post-diapause development also helps to condense the emergence of flies in the spring. Hence, an accurate forecast of the time of spring attack by populations of flies similar to those at Wellesbourne should be possible.This study was financed partly by the Commission of the European Communities as CEC Contract No. 0771.