Platelet-reactive sites in collagen. Collagens I and III possess different aggregatory sites.

Collagen type III possesses a highly reactive platelet-aggregatory site at a locus which in type I is essentially inactive whilst the latter collagen possesses reactive sites absent in type III. It is proposed that platelet aggregation by collagen involves the sequence GK[or R]PG(EY)GPK[or R]G(EY) or, less favourably, GPK[or R]G(EY)G(XY)GK[or R]PG(EY), one basic residue acting in combination with the second in an adjacent alpha-chain.     

Metabolism and excretion of peptide leukotrienes in the anesthetized rat

The metabolism and excretion of the peptide leukotrienes C4, D4, E4 and N-acetylleukotriene E4 have been studied in the anesthetized rat. The intravenous administration of [3H]leukotriene C4 (2.6 X 10(-11) mol/kg) showed a rapid clearance of radioactivity from the blood and a time-related biliary excretion, recovering 69 +/- 1.6% (n = 6) over 60 min. Less than 1% of total radioactivity was recovered in the urine over the same time period. Similarly, the intravenous administration of [3H]leukotriene D4 (2.5 X 10(-11) mol/kg), [3H]leukotriene E4 (2.5 X 10(-11) mol/kg) and N-acetyl[3H]leukotriene E4 (2.1 X 10(-11) mol/kg) showed a 62 +/- 7.5% (n = 4), 52 +/- 1.5% (n = 4) and 37 +/- 4.6% (n = 5) biliary recovery of radioactivity, respectively, after 60 min. Examination of bile identified leukotriene D4 and N-acetylleukotriene E4 as the main products, although substantial radioactivity, which probably represents unidentified polar products, was present at the solvent fronts of the reverse-phase HPLC. Time course studies indicated a relatively rapid conversion of leukotriene C4 to leukotriene D4, while leukotriene D4 metabolism appeared to be much slower. Leukotriene E4 was a minor product, suggesting that the N-acetylation process is rapid. Incubation of [3H]leukotriene C4 in rat plasma and whole blood in vitro resulted in a slow conversion of leukotriene C4 to leukotriene D4 and leukotriene E4 only. These data suggest that the majority of the leukotriene metabolism and excretion in vivo in the anesthetized rat occurs predominantly in the hepatic system. We conclude that this model is suitable for the measurement of in vivo production of peptide leukotrienes.     

Synthesis of [2-3H-ethyl]S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and its use in covalent-binding studies.

Metabolism of S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) yields chlorofluorothioacetyl fluoride, which reacts with cellular proteins to form stable lysine adducts. Little is known about the subcellular localization of these protein adducts or about their role in CTFC-induced nephrotoxicity. A method for the synthesis of CTFC and other cysteine S-conjugates labeled with 3H at the S-alkyl or S-alkenyl position would be useful in studies of S-conjugate metabolism and toxicity. Reaction of L-cysteine, chlorotrifluoroethene, 1,8-diazabicyclo[5.4.0]undec-7-ene, and 3H-labeled water followed by repeated crystallization yielded radiochemically pure [3H]CTFC (235 mg, 20% yield; sp act 1.07 x 10(9) Bq/mmol), which was identical to CTFC by TLC, 1H NMR, and 19F NMR. 3H NMR revealed a doublet of triplets at 6.5 ppm with geminal and vicinal T-F couplings of 51.5 and 6.0 Hz, respectively, consistent with the proposed structure. When 2H-labeled water was used, [2H]CTFC was formed, and its structure was confirmed by 1H and 19F NMR, FAB-MS, and TLC. Analysis of renal and hepatic subcellular fractions of rats given 1, 10, or 100 mumol/kg [3H]CTFC showed a dose-dependent binding of 3H-containing metabolites to liver and kidney proteins.     

Cellular Choline and Glycine Betaine Pools Impact Osmoprotection and Phospholipase C Production in Pseudomonas aeruginosa

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used (13)C nuclear magnetic resonance ((13)C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology.     

The genetic structure of Australasian green turtles (Chelonia mydas): exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Phi = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.     

Efficiency clustering for low-density microarrays and its application to QPCR

Background  

Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency.     

Evidence of in-vivo omega-oxidation of peptide leukotrienes in the rat: biliary excretion of 20-CO2H N-acetyl LTE4

In a previous study in our laboratory it was observed that after [3H] LTC4 administration (luCi/kg i.v.) to the anesthetized rat, significant amounts of injected radioactivity (approximately 25%) were associated with previously unidentified biliary polar metabolite(s). In the present study we describe the isolation and characterization of the predominant polar metabolite. Rats were injected with synthetic LTC4 (20 microgram/kg i.v.) and bile collected over 30 min. After extraction and purification (2 step RP-HPLC procedure), the retention time of the metabolite was compared (plus coinjections) and found to be identical with synthetic 20-CO2H N-Ac LTE4 in two RP-HPLC systems. Also, the UV spectrum of the biologically derived metabolite was compared and found identical to the synthetic material, giving a characteristic conjugated triene absorption in the UV with a max of 281 nm and shoulders at 270 and 290 nm. Further, the trimethyl ester derivative of the metabolite showed identical chromatographic behaviors in 2 reverse and 2 normal phase HPLC systems compared with synthetic 20-CO2H N-Ac LTE4 trimethyl ester. We conclude omega-oxidation of peptide leukotrienes occurs in the rat and that 20-CO2H N-Ac LTE4 is an in vivo product of LTC4 metabolism.