Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate.

The minimal substrate for the 2 microns circle site-specific recombinase FLP consists of a nearly perfect 13-base-pair dyad symmetry with an 8-base-pair core. By using a series of chemically synthesized FLP substrates in in vitro FLP recombination and FLP-binding assays, we have identified four positions within each of the symmetry elements that are important contact points for the FLP protein. Furthermore, the binding and recombination data provide evidence for cooperativity between the two symmetry elements of a substrate and between the symmetry elements of two partner substrates during FLP recombination.     

Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5''-p-fluorosulphonylbenzoyladenosine.

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). Optimal inactivation by 5'-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5'-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5'-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5'-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5'-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5'-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5'-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.     

Inducing sporulation in Alternaria solani II. Effect of light

Among the three different light sources viz. incandescent electric light, infra-red light and sunlight, only sunlight was effective in inducing sporulation in petri dish cultures ofA. solani. The intensity of sporulation depended upon the age and growth stage of the cultures, duration and number of exposures to light and the presence or absence of a sporulating zone in the culture. Maximum sporulation was obtained in the case of 6 days old partially grown cultures by inducing the formation of sporulating zone which appeared in 24 hours after every exposure of 60 minutes to sunlight.     

Effect of zinc on increasing oxygen affinity of sickle and normal red blood cells

We have hypothesized a state of zinc deficiency in sickle cell disease (SCD). This could at least partially explain the growth problems, hypogonadism, and slow healing leg ulcers associated with SCD. Preliminary findings revealed abnormally low red blood cell zinc levels in 10 of 16 patients studied. Before suggesting zinc supplementation in SCD we thought it important to look at the effect of zinc on red cell metabolism and function. It was found that zinc chloride added to normal and SCD blood to a final concentration of 1.5 × 10^?3 M caused a left-shift of the blood oxygen affinity curve (increased oxygen affinity) varying from 1.5 to 3.5 mm Hg change in half saturation (p50). This curve shifting property has important implications for SCD since recent work with cyanate suggests that such shifts are very beneficial in treatment of SCD. Thus zinc supplementation in SCD, in addition to its potential role in correcting wound healing and growth problems, may have a beneficial effect on the basic pathological process. Data are given which suggest that zinc and 2, 3-diphosphoglycerate may not be competing for the same site on the hemoglobin molecule.     

Dry-matter production and recovery of fertilizer nitrogen by rice as affected by nitrification retarders ‘N-Serve’ and ‘AM’

Summary In a pot-culture experiment simulating semi low-land rice field conditions 5 to 11 per cent increase in dry matter yield and 27 to 43 per cent increase in recovery of applied N was obtained by the use of N-Serve and AM nitrification retarders.Although the term frequently used is 'nitrification inhibitors, the term nitrification retarders is proposed since under field conditions these chemicals only partially control the nitrification.Trade name of The Dow Chemical Company, Midland, Michigan, U.S.A. for 2-chloro-6-(trichloromethyl) pyridine.Trade name of Toyo Koatsu Industries, Inc., Tokyo, Japan for 2-amino-4chloro-6methyl pirimidine.     

Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin

The structure of the elastin polypentapeptide, poly(VPGVG), was studied by nuclear Overhauser effect experiments using perdeuterated Val1 and Val4 samples under the condition where intermolecular interactions are absent. More extensive interaction was found between the Val1 gamma CH and Pro2 beta CH protons than between the Val4 gamma CH and Pro2 beta CH protons. The Val1 gamma CH3-Pro2 beta CH interaction does not occur within the same pentamer as previously shown experimentally and as expected from steric considerations. The results are incompatible with the presence of a random chain network in poly(VPGVG) at room temperature but are readily explicable in terms of interturn interactions in a beta-spiral structure. More specifically, the results indicate that the beta-spiral conformation with 2.9 pentamers/turn is more prevalent than that with 2.7 pentamers/turn. Using conformations developed by molecular mechanics calculations, molecular dynamics simulations were carried out to compare the relative energies of these two variants of this class of beta-spiral structures. It was found in vacuo that the structure with 2.9 pentamers/turn is indeed more stable than that of 2.7 pentamers/turn by approximately 1 kcal/mole-pentamer.     

Emerging role of lipids of Candida albicans, a pathogenic dimorphic yeast.

It is clear that C. albicans lipids have gained tremendous importance in recent years. In addition to being a barrier for entrance of various metabolites, it also provides the site of action for the synthesis of enzyme(s) involved in cell wall morphogenesis and antifungal action. While alterations in lipid composition during a yeast to mycelia transition have been observed, in most of the studies, lipid fluctuations reported could have been due to various environmental factors involved in the induction of morphogenesis [4,5]. A clear understanding of lipid biosynthesis and metabolic blocks due to antifungal action is likely to shed further light on selective interactions of antifungals. Despite the multifacet role of lipids in various functions of this pathogenic yeast, their exact involvement is poorly understood. The situation is little better with regard to ergosterol and its metabolism. Ergosterol is, indeed, important for anti-candidal activity and appears to be involved in the morphogenesis of C. albicans. The fluctuation in phospholipid composition have led to altered properties of plasma membrane namely, membrane fluidity, transport activities and drug sensitivity, which suggest that-a critical level of individual phospholipid is important for proper functioning of the plasma membrane. What the exact role is of individual phospholipid is far from clear. Many unanswered questions relating to the role of PI and sphingomyelin in signal transduction, involvement of phospholipases in the maintenance of phospholipid composition, and role of lipid transfer proteins in assembly and asymmetry of lipids are some aspects which merit further work.