Rhythmic patterns in phagocytosis and the production of reactive oxygen species by zebrafish leukocytes

Multiple components of vertebrate immune systems have been shown to exhibit circadian fluctuations. While the zebrafish is currently generating a wealth of information on the molecular pacemakers that may control circadian rhythms, there have been no reports of rhythmic activity in zebrafish leukocytes. In this study, we found that phagocytosis and the production of reactive oxygen species by zebrafish leukocytes varied significantly throughout twenty-four hour periods. A distinct peak in cellular ROS levels occurred before dawn, while the kinetics of respiratory burst responses were least rapid at this time of day. Phagocytosis of E. coli peaked late in the day, whereas there was no daily variation in phagocytosis of S. aureus. As seen in other species, the number of bacteria ingested per cell peaked during the night. These data provide direct evidence of rhythmic immune system activity, and demonstrate that zebrafish can be a valuable model in which to study the relationships between circadian gene expression, systemic pacemakers, and the activity of vertebrate immune system cells.     

Machine-learning approaches for classifying haplogroup from Y chromosome STR data

Genetic variation on the non-recombining portion of the Y chromosome contains information about the ancestry of male lineages. Because of their low rate of mutation, single nucleotide polymorphisms (SNPs) are the markers of choice for unambiguously classifying Y chromosomes into related sets of lineages known as haplogroups, which tend to show geographic structure in many parts of the world. However, performing the large number of SNP genotyping tests needed to properly infer haplogroup status is expensive and time consuming. A novel alternative for assigning a sampled Y chromosome to a haplogroup is presented here. We show that by applying modern machine-learning algorithms we can infer with high accuracy the proper Y chromosome haplogroup of a sample by scoring a relatively small number of Y-linked short tandem repeats (STRs). Learning is based on a diverse ground-truth data set comprising pairs of SNP test results (haplogroup) and corresponding STR scores. We apply several independent machine-learning methods in tandem to learn formal classification functions. The result is an integrated high-throughput analysis system that automatically classifies large numbers of samples into haplogroups in a cost-effective and accurate manner.     

Formation of prototrophs in mixtures of two auxotrophic mutants of Serratia marcescens HY by a transducing bacteriophage produced by some auxotrophs

Summary Prototrophs arising in mixtures of two auxotrophs of Serratia marcescens strain HY are caused by a filtrable agent produced by one of the partners. 3. donors of this filtrable agent, HY/thyl, HY/ade11, and HY/thr2, were found among 16 auxotrophs, strain HY/thyl producing the agent of the highest activity. The prototrophic wildtype HY is not a donor. The agent does not enhance the growth of the auxotrophic recipient bacteria on minimal medium, therefore the increase in prototophs is not due to more spontaneous mutations. Dilution experiments showed that the reaction of one agent particle with a recipient-cell can cause a prototroph and that the number of recipient cells is not the limiting factor of prototroph formation.When the donor of a filtrable agent contained (besides the agent-inducing thyl-auxotrophy) a second auxotrophy of the same type as the recipient the relative frequency of prototrophs formed was much lower than that one produced by the HY/thyl filtrate. This indicates an influence of pseudoallelism of the markers on prototroph formation as would be expected by transfer of genetic material.The filtrable agent is non-dialyzable, precipitates by ammonium sulfate and is resistant to unspecific phosphodiesterase. When the filtrate was centrifuged in a CsCl density-gradient (24 hours at 35000 rpm) a band occurred at a density of 1.497 g/cm³ which contained the activities for prototroph and plaque formation. It contained also much material with an UV-absorption spectrum typical of phages. Electron micrographs revealed this material to consist of phage particles with hexagonal heads of 50 m diameter and a very short tail. These particles were named y phage.One strain, AX, of Serratia marcescens (among 47 tested) gave small, turbid plaques with filtrates from the known donors but not from other auxotrophs or HY. The plaque titer of y phage on AX was about the same as the transduction to prototrophy of HY/leu27. Phage y is a general-transducing bacteriophage of Serratia marcescens HY since 13 auxotrophic markers of strain HY and 10 of strain AX could be transduced. The low e.o.p. of y phage produced by HY/thyl, may therefore not be due to restriction by the AX cells but may indicate that this y phage is defective.     

Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and photosynthetic cell cultures

The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8,300 peptides were identified with high confidence from at least one subcellular fraction from either cell culture. These peptides were derived from 1,514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single subcellular fraction and their localization was compared to in silico predictions. However, the majority of proteins were observed in multiple subcellular fractions, and the most likely subcellular localization for these proteins was investigated using a Z-score analysis of estimated protein abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.     

Lectin Binding to Radopholus citrophilus and R. similis Proteins

Lectin-binding glycoproteins in seven populations of two burrowing nematode sibling species were probed with five different biotinylated lectins on Western blots, and differences were correlated with nematode ability to parasitize citrus and to overcome citrus rootstock resistance. Banding patterns of molecular weight standards were fit best by an exponential decay function, and a predictive equation was used to estimate molecular weights (r² = 0.999). A band (131 kDa) that labeled with the lectin Concanavalin A (Con A) occurred in extracts from cuticles and egg shells of populations of Radopholus citrophilus that parasitize citrus. Wheat germ agglutin labeled a band (58 kDa) in aqueous homogenates of populations that reproduce in roots of citrus rootstock normally resistant to burrowing nematodes. The two sibling species R. citrophilus and R. similis were distinguished by a high molecular weight Con A-labeled band (608 kDa) from cuticle and egg shells. Probing blots with the lectin Limulus polyphemus agglutinin indicated that each population contained a band (12-16 kDa) specifically inhibited by the addition of 25 mM neuraminic acid, suggesting that glycoproteins with sialic acid moieties are present in burrowing nematodes.     

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

Experimental radio-osteonecrosis in Rhesus macaque jaws; therapeutic irradiation dose effect on dental extraction wound healing

Varying degrees of osteonecrosis result from molar extractions or other oral surgical procedures in rhesus macaques before, during, or after the administration of 6000 rad (the equivalent of human therapeutic dose levels) via either 240 kVp x-ray or cobalt 60 irradiation to the jaw areas subjected to surgery. The effects of various experimental time-dose-wound combinations on the level of impairment of wound-healing, interruption of osteogenesis, and the nature of the response of the irradiation-compromised tissues are covered in this report.