Purification and properties of the membrane-bound CDP-diglyceride synthetase from Escherichia coli

The enzyme CDP-diglyceride synthetase (CTP: phosphatidate cytidylyltransferase; EC 2.7.7.41) has been purified to 90% homogeneity from Escherichia coli cells that overproduce the enzyme 50-fold through the use of recombinant DNA technology. The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein. Apparent physical effects of EDTA on the enzyme were also utilized in the purification. The enzyme has an apparent minimum subunit mass of 27,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino acid composition of the protein was determined, and it correlates well with the theoretical protein product of the cds gene, the sequence of which is reported in the accompanying paper (Icho, T., Sparrow, C. P., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 12078-12083). The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. As previously suggested on the basis of studies using partially purified preparations, the enzyme mechanism is sequential, and computer-calculated kinetic constants are reported herein. The substrate specificity of the enzyme is also investigated. This is the first time this enzyme has been purified to homogeneity from any source, despite the fact that it is essential for phospholipid biosynthesis in all organisms.     

Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope

Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XI. A PHYSODERMA ON AGROPYRON REPENS

Sparrow , Frederick K. (U. Michigan, Ann Arbor), Joyce E. Griffin , and Robert M. Johns . Observations on chytridiaceous parasites of phanerogams. XI. A Physoderma on Agropyron repens. Amer. Jour. Bot. 48(9): 850–858. Illus. 1961.—A Physoderma has been found in Michigan on Agropyron repens, which produces dark-brown, non-erumpent streaks up to 20 mm (usually 4–6 mm) in length by 0.5 mm in width and very local discoloration of the leaf blades and sheaths. There is no systemic infection, and plants parasitized are normal in size and bear flowers. Resting spores upon germination produce zoospores which may develop either into the endobiotic, poly-centric, resting spore-bearing thallus or into monocentric, epibiotic, ephemeral sporangia. The latter bear either colorless or orange globuled zoospores. These zoospores may give rise to new epibiotic sporangia, or, apparently without fusing in pairs, to the endobiotic system. Comparison with other congeneric fungi reported on Agropyron repens reveals the Michigan fungus to be distinct from them. Whether it can be embraced within Physoderma graminis (Büsgen) de Wild. or other described taxa on grasses awaits the results of future cross-inoculation studies.     

Binding of a high reactive heparin to human apolipoprotein E: identification of two heparin-binding domains

Ligand-blotting and dot-blotting procedures were used to investigate the binding of [125I]-heparin to apolipoprotein E, its thrombin fragments E22 (residues 1-191) and E12 (residues 192-299), and to nine apolipoprotein E synthetic fragments. E22 and E12 bound [125I] heparin indicating multiple heparin-binding domains. Synthetic peptides of apoE corresponding to residues 129-169, 139-169, and 144-169, but not 148-169, bound [125I] heparin suggesting that residues 144-147 (Leu-Arg-Lys-Arg) in E22 are important for binding. Peptide 202-243 and 211-243 but not 219-243 bound [125I] heparin suggesting that residues 211-218 (Gly-Glu-Arg-Leu-Arg-Ala-Arg-Met) comprise a portion of the E12 heparin-binding domain.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

Comparison of Respiratory Disease Prevalence among Voluntary Monitoring Systems for Pig Health and Welfare in the UK

Surveillance of animal diseases provides information essential for the protection of animal health and ultimately public health. The voluntary pig health schemes, implemented in the United Kingdom, are integrated systems which capture information on different macroscopic disease conditions detected in slaughtered pigs. Many of these conditions have been associated with a reduction in performance traits and consequent increases in production costs. The schemes are the Wholesome Pigs Scotland in Scotland, the BPEX Pig Health Scheme in England and Wales and the Pig Regen Ltd. health and welfare checks done in Northern Ireland. This report set out to compare the prevalence of four respiratory conditions (enzootic pneumonia-like lesions, pleurisy, pleuropneumonia lesions and abscesses in the lung) assessed by these three Pig Health Schemes. The seasonal variations and year trends associated with the conditions in each scheme are presented. The paper also highlights the differences in prevalence for each condition across these schemes and areas where further research is needed. A general increase in the prevalence of enzootic pneumonia like lesions was observed in Scotland, England and Wales since 2009, while a general decrease was observed in Northern Ireland over the years of the scheme. Pleurisy prevalence has increased since 2010 in all three schemes, whilst pleuropneumonia has been decreasing. Prevalence of abscesses in the lung has decreased in England, Wales and Northern Ireland but has increased in Scotland. This analysis highlights the value of surveillance schemes based on abattoir pathology monitoring of four respiratory lesions. The outputs at scheme level have significant value as indicators of endemic and emerging disease, and for producers and herd veterinarians in planning and evaluating herd health control programs when comparing individual farm results with national averages.