Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions

The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100 degrees C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein.     

Prevention and therapy of experimental autoimmune neuritis by an antibody against T cell receptors-alpha/beta.

The mAb R73 directed to the TCR-alpha/beta of rat lymphocytes was tested for its therapeutic potential during the effector phase of experimental autoimmune neuritis (EAN) in Lewis rats. EAN can be actively induced by immunization with bovine peripheral nerve myelin, bovine P2 protein, or a peptide containing its neuritogenic epitope and serves as a model of the human Guilain-Barré syndrome. Adoptive transfer of activated P2-specific T lymphocytes also produces the monophasic disease (AT-EAN) characterized by inflammation and demyelination of peripheral nerves and highlights the central role of T lymphocytes in the pathogenesis of EAN. A single administration of the mAb R73 immediately after injection of activated P2-specific T line cells completely prevented the development of clinical and electrophysiologic signs of EAN in most animals and greatly alleviated the disease in the others. In further experiments mAb R73 was applied after the appearance of first clinical signs of EAN actively induced by immunization with a neuritogenic peptide or bovine peripheral nerve myelin. In both cases the anti-TCR-alpha/beta mAb reversed clinical signs of EAN and prevented the development of peripheral nerve dysfunction. In vivo and in vitro data suggest that impairment of Ag recognition and T cell function by occupancy of the TCR and R73-induced TCR-modulation rather than depletion of TCR-alpha/beta-bearing lymphocytes is the decisive mechanism underlying suppression of EAN that is apparent already within 48 h of the first R73 injection.     

Role of catecholamines in hypotensive response to dieting.

The effect of dieting on blood pressure and catecholamine metabolism was assessed in 11 normotensive obese women by providing first a weight-maintenance regimen high in carbohydrate and then a low-energy diet. All dietary constituents other than carbohydrate were maintained constant throughout the 18-day study. The low-carbohydrate diet led within 48 hours to a 41% fall in the urinary output of 4-hydroxy-3-methoxy mandelate and a significant fall in systolic and diastolic blood pressure. Plasma noradrenaline concentrations also fell and the hypotensive effect of the diet continued despite a maintained total body sodium. Thus the fall in blood pressure appeared to be mediated by changes in catecholamine metabolism independent of sodium intake. This may explain both the usefulness of weight reduction in hypertensive patients and the fainting that occurs in some normotensive obese subjects taking slimming regimens low in carbohydrate.     

Electrophoretic studies of several hydrolytic enzymes in relation to the cold tolerance of alfalfa.

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to determine the characteristics and relationships of several hydrolytic enzymes to cold tolerance.Soluble enzymatic proteins, extracted from lyophilized crown and root tissues with three different solvents, were separated by polyacrylamide disc-gel electrophoresis and evaluated on the basis of equal dry weights of tissue and equal quantities of protein.Gels assayed for amylases, acid phosphatases, esterases, leucine aminopeptidases, and adenosine triphosphatases exhibited mainly quantitative differences in isoenzymes depending upon extractant, cultivar, and environmental differences. The qualitative differences detected were generally due to differential solubilities of isoenzymes in the three extractants and, to a lesser extent, were related to environmental, cultivar, or stability differences.While activities of esterases, acid phosphatases, and leucine aminopeptidases increased in winter samples, as soluble protein increased, only slight decreases in these enzymes occurred during dehardening. Conversely, activities of amylases were slightly lower in winter samples than in the other samples, and adenosine triphosphatase activity decreased in the most coldtolerant sample.The measured levels of total nonstructural carbohydrate, total soluble sugar, and starch indicated differences between cultivars in starch-sugar conversion. Further, the differential heat stabilities of the isoamylases also provided some information as to the nature of “protected activity” of diastatic enzymes.Differential cryostabilities of peptidases and adenosine triphosphatases detected between cultivars and environments also demonstrated the influence of the extraction medium in maintaining enzyme activity, and these observations may be important to an understanding of cold tolerance in alfalfa. The obvious speculations regarding enzyme stability and the factors involved as related to the cold tolerance of alfalfa require further examination.     

Sterically hindered disulfide bridges in cystine diketopiperazine, cysteinyl-cysteine disulfide and derivatives.

L-Cystine diketopiperazine (1), L-cysteinyl-cysteine disulfide -HCl (2), L-cysteinyl-cysteine disulfide methyl ester -HCl (3), and t-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) are investigated by CD, ultraviolet, 13C NMR, infrared and laser Raman spectroscopy. The temperature dependence of the 13C NMR signals of 1 reveals an exceptionally high energy barrier of deltaGNo. = 15.8 +/- 0.2 kcal/mol for the reversible change in helicity of the inherently dissymmetric disulfide bridge of 1. The P-helical diastereomer predominates in dimethyl-sulfoxide at 25 degrees C, with 80-85% of the molecules having this configuration. The Cotton effects of 1 are larger and show smaller temperature coefficients than the conformationally more mobile cystine compounds 2 and 3. After dissolving crystals of 1 in 95% ethanol there is a time-dependent decrease of the ellipticity of the negative Cotton effect at 225 nm, indicating a conformational change in going from crystal to solution. Besides 1, 2 and 3 are at present the only known examples of cystine derivatives with C-S-S-C torsional angles around 90 degrees, which do not exhibit optical activity in the long wavelength disulfide absorption, as is predicted for 1 from the Linderberg-Michl model. At 305 nm a new weak Cotton effect was discovered for 1. The solvent dependence of the CD spectra is discussed and the infrared and Raman spectra are assigned.     

Kinetic Monte Carlo simulation for the striation distribution of void defects in Czochralski silicon growth

Unique crystal-originated pit (COP) distribution, similar to a striation pattern, is well matched with the oxygen profile in experimental analysis. It shows the strong relationship between oxygen concentration and COP distribution. In this paper, we study the generation of void defects and the relationship between interstitial oxygen and vacancy using the kinetic lattice Monte Carlo (KLMC) method. The KLMC method has been applied extensively in various forms to the study of micro-defects in silicon wafers. It explained well the formation of void defects such as vacancy–oxygen complex and vacancy–vacancy complex. The formation of clusters is strongly affected by oxygen concentration, which showed the relationship between COP distribution and oxygen concentration. The unique COP distribution could be correctly explained with KLMC results, and this kind of meso-scale results has not yet been reported.     

Staphyloferrin A: a structurally new siderophore from staphylococci

Two ferric ion-binding compounds, designated staphyloferrin A and B, were detected in the culture filtrates of staphylococci grown under iron-deficient conditions. Staphyloferrin A was isolated from cultures of Staphylococcus hyicus DSM 20459. The structural elucidation of this highly hydrophilic, acid-labile compound revealed a novel siderophore, N2,N5-di-(1-oxo-3-hydroxy-3,4-dicarboxybutyl)-D-ornithine, which consists of one ornithine and two citric acid residues linked by two amide bonds. The two citric acid components of staphyloferrin A provide two tridentate pendant ligands, comprising of a beta-hydroxy, beta-carboxy-substituted carboxylic acid derivative, for octahedral metal chelation. The CD spectrum of the staphyloferrin A ferric complex indicates a predominant A configuration about the ferric ion center. The uptake of ferric staphyloferrin A by S. hyicus obeys Michaelis-Menten kinetics (Km = 0.246 microM; vmax = 82 pmol.mg-1.min-1), indicating active transport of this siderophore. The staphyloferrin A transport system is different from that of the ferrioxamines as shown by an antagonism test. Production of staphyloferrin A is strongly iron-dependent and is stimulated by supplementation of the medium with either D- or L-ornithine. DL-[5-14C]ornithine was incorporated into staphyloferrin A, demonstrating that ornithine is an intermediate in staphyloferrin A biosynthesis.     

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.     

Cardiovascular disease mortality in Canada.

During the past two decades approximately one half of all deaths in Canada were due to cardiovascular diseases. Ischemic heart disease and cerebrovascular disease caused more than 60% and 20% of those deaths respectively. The mortality rates for ischemic heart disease in males increased slightly until 1965 and then dropped substantially, whereas the rates for females, which were declining at least since the early 1960s, accelerated in their decline. As a consequence, the rates for males remain almost twice as high as those for females. The reductions were initially observed in males 25 to 34 years old and in all age groups of females, but became apparent in a wider range of ages in the second period reviewed (1969 through 1977). The mortality of cerebrovascular disease has gradually diminished for both sexes since the 1950s, but the decline has been more pronounced among females, who originally had the higher rate. Marked geographic differences in mortality rates still exist in Canada despite the decline in death rates for both ischemic heart disease and cerebrovascular disease in all regions of the country. Surprising regional differences in times of onset of these declines have been demonstrated. For ischemic heart disease Ontario maintains the highest and the Prairies the lowest mortality rates. Quebec, despite a sustained decline, still ranks third, while the Pacific region shows the second-lowest rates in the country. The Atlantic region showed the lowest rates of decline in the period reviewed. The reduction in the mortality of ischemic heart disease in Canada (16.4% between 1969 and 1977) must be considered real for a variety of reasons. Direct evidence is not available to elucidate whether the reduction is the consequence of reduced incidence, increased survival or a combination of the two factors. The potential role of various factors that may have contributed to this decline is briefly discussed in this article.