Effect of Salinity on Rhizobium Growth and Survival

The microbiological metabolism of warfarin was examined as a model of metabolism in higher organisms, including humans, and to determine the chirality of microbial reductases for application in organic synthesis. Nineteen cultures were examined based on their reported abilities to reduce ketonic substrates, and several were shown to catalyze the desired reaction. Nocardia corallina (ATCC 19070) exhibited complete substrate and product stereoselectivity as it reduced S-warfarin to the corresponding S-alcohol. Arthrobacter species (ATCC 19140) exhibited marked substrate and complete product stereoselectivity since S-warfarin, and to a lesser extent R-warfarin, were reduced to the corresponding S-alcohols. These reductions parallel those reported to occur in mammalian species.     

Effect of pH,mono- and divalent cations on the mixing of phosphatidylglycerol with phosphatidylcholine. A monolayer (π, ΔV) and fluorescence study

The mixing of various molecular species of phosphatidylglycerol and phosphatidylcholine differing in their acyl chain lengths has been studied both in monolayers (π, Δ$\text{V}$), and in water dispersions (fluorescence polarization) with varying pH and ionic strength of the aqueous phase and in the presence of the divalent cations Mg²⁺ and Ca²⁺. In dilauroylphosphatidylglycerol/dipalmitoylphosphatidylcholine mixtures, both in monolayers and in water dispersions, no phase separation was detected at pH 2.9 where phosphatidylglycerol was protonated. With dipalmitoylphosphatidylglycerol/dipalmitoylphosphatidylcholine mixtures, in monolayers and at the same pH, no phase separation was detected for surface pressures below $\text{π = 40}\text{mN}\text{·}\text{m}{}^{\mathrm{?1}}$. In monolayers, and under ionic conditions such that phosphatidylglycerol was ionized (pH 5.6, 10 mM NaCl) miscibility was observed with dilauroylphosphatidylglycerol and dipalmitoylphosphatidylcholine and also with dipalmitoylphosphatidylglycerol and dilauroylphosphatidylcholine. Varying the ionic strength did not alter the miscibility of these lipids. The divalent cations Mg²⁺ and Ca²⁺ did not modify that of dilauroylphosphatidylglycerol with dilauroylphosphatidylcholine or with dipalmitoylphosphatidylcholine. Both in monolayers and in water dispersions, dipalmitoylphosphatidylglycerol and dilauroylphosphatidylcholine appeared to be at least partly miscible, in the presence of magnesium. Only in the presence of calcium and at high surface pressure might the monolayer data account for phase separation between these two lipids. The data presented demonstrate the existence of strong cohesive forces between phosphatidylcholine and phosphatidylglycerol with a marked influence of the former on the physical state of the latter. From an analysis of the Δ$\text{V}$ data, it is suggested that intrafacial hydrogen bonds may play a significant role in stabilizing phosphatidylcholine/phosphatidylglycerol mixtures.     

Studies on dehydro-l-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro-l-ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-(l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-(l-threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-l-threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

Comparative titration of arginyl residues in purified D-β-hydroxybutyrate apodehydrogenase and in the reconstituted phospholipid-enzyme complex

In order to titrate and understand the role of arginyl residues of D-β-hydroxybutyrate dehydrogenase, arginyl specific reagents: butanedione, 1,2-cyclohexanedione and phenylglyoxal were incubated with three different forms of the enzyme; native enzyme (inner mitochondrial membrane bound), purified apoenzyme (phospholipid -free) and phospholipid-enzyme complex (reconstituted active form).After complete inactivation of the enzyme by [¹⁴C]-phenylglyoxal, the number of modified arginyl residues was different: one with the lipid-free apoenzyme and three with the phospholipid-enzyme complex, suggesting a conformational change of the enzyme triggered by the presence of phospholipids.After exhaustive chemical modification either of the apoenzyme or of the phospholipid-enzyme complex with [¹⁴C]-phenylglyoxal, four arginyl residues were titrated indicating that these residues are located in the hydrophilic part of the enzyme, not interacting with phospholipids.Reconstituted enzyme inactivated by butanedione could no longer bind a pseudosubstrate (succinate) which indicates that an arginyl residue is involved in the enzyme-substrate complex formation.The values of second order rate constants of D-β-hydroxybutyrate dehydrogenase inactivation by butanedione and 1,2-cyclohexanedione were unchanged with the three enzyme forms, suggesting that phospholipids are not involved in the substrate binding mechanism.     

High-performance reversed-phase chromatographic mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides.

Xyloglucan oligosaccharides from cotton cell walls and tamarind seeds were derivatized with 2-aminopyridine and subsequently separated by reversed-phase chromatography (r.p.c.) using an octadecylsilyl silica stationary phase and aqueous-organic eluents with 0.01% (v/v) trifluoroacetic acid. The chromatographic behavior of the 2-pyridylamino derivatives of xyloglucan oligosaccharides was examined under a wide range of elution conditions, including gradient steepness and shape, initial acetonitrile concentration in the eluent, and pore size of the r.p.c. packings. Relatively steep acetonitrile gradients resulted in poor resolution of the different xyloglucan fragments, which is believed to be the result of acetonitrile-induced conformational changes. Under these circumstances the elution order of the derivatized xyloglucan oligosaccharides was such that the smaller fragments eluted from the column before the larger ones. R.p.c. packing with a 70-A pore size necessitated relatively high acetonitrile concentration in the eluent when compared with 300-A stationary phase. The r.p.c. mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides was best achieved when both a wide-pore octadecyl-silyl silica stationary phase and a shallow gradient with consecutive linear segments of increasing acetonitrile concentration in the eluent were employed. This combination yielded rapid r.p.c. maps of the xyloglucan fragments from different sources with high separation efficiencies and concomitantly high resolution. The effects of the nature of the sugar residues in the xyloglucan oligomers and their degree of branching on r.p.c. retention and selectivity are also highlighted.     

Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

Summary A characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.     

DNA amplification fingerprinting of bacteria

Summary We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng·l^–1 and high MgCl₂ levels was especially important for reproductibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.Correspondence to: G. Caetano-Anollés     

Aphid Vectors of Barley Yellow Dwarf Virus in West-Central Morocco

The ability of seven aphid species, collected in west-central Morocco, to transmit barley yellow dwarf virus (BYDV) was determined. Aphids were either collected from grasses showing symptoms of BYDV infection or were allowed acquisition access to plants infected with a PAV-like isolate of BYDV before transfer to oat test plants. BYDV transmission by six of the seven aphid species was confirmed by ELISA test; only Melanaphis donacis failed to transmit. The six newly defined BYDV vector species brings the total known to occur in Morocco to ten.     

Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR

Hydrogen exchange and two-dimensional nuclear magnetic resonance (2D NMR) techniques were used to characterize the structure of oxidized horse cytochrome c at acid pH and high ionic strength. Under these conditions, cytochrome c is known to assume a globular conformation (A state) with properties resembling those of the molten globule state described for other proteins. In order to measure the rate of hydrogen-deuterium exchange for individual backbone amide protons in the A state, samples of oxidized cytochrome c were incubated at 20 degrees C in D2O buffer (pD 2.2, 1.5 M NaCl) for time periods ranging from 2 min to 500 h. The exchange reaction was then quenched by transferring the protein to native conditions (pD 5.3). The extent of exchange for 44 amide protons trapped in the refolded protein was measured by 2D NMR spectroscopy. The results show that this approach can provide detailed information on H-bonded secondary and tertiary structure in partially folded equilibrium forms of a protein. All of the slowly exchanging amide protons in the three major helices of native cytochrome c are strongly protected from exchange at acid pH, indicating that the A state contains native-like elements of helical secondary structure. By contrast, a number of amide protons involved in irregular tertiary H-bonds of the native structure (Gly37, Arg38, Gln42, Ile57, Lys79, and Met80) are only marginally protected in the A state, indicating that these H-bonds are unstable or absent. The H-exchange results suggest that the major helices of cytochrome c and their common hydrophobic domain are largely preserved in the globular acidic form while the loop region of the native structure is flexible and partly disordered.     

Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines

Summary All the five enzymes of urea synthesis and the formation of urea in vitro can already be demonstrated in human liver as early as the 9th week of fetal development. At this stage the activity of carbamoyl phosphate synthetase is the highest, whereas that of ornithine carbamoyltransferase is the lowest as compared to those in the adult. The kinetic parameters of the urea cycle enzymes are the same in fetal liver as in adult liver, except that the Km values of ornithine carbamoyltransferase for L-ornithine are 3.5 mM and 0.42 mM in the fetus and in adult liver, respectively.Urea formation in vivo seems to begin in the second half of fetal life, and a gradual increase can be detected in the activity of the enzymes of urea synthesis. The activity of ortnithine decarboxylase, the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase, however, changes in the opposite direction.The concentration of carbamoyl phosphate and aspartate remains constant, but that of ornithine gradually decreases during ontogenesis. The ornithine, carbamoyl phosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.