Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua

A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.     

DNA core ionization and cell inactivation

Whether inner-shell ionizations of DNA atoms, called core ionizations, are critical events for cell inactivation by ionizing radiations such as 100 keV electrons and gamma rays has been investigated. The number of core ionizations in DNA atoms per gray of the two types of radiations is calculated from various Monte Carlo track simulations. The probability that a core ionization leads to cell inactivation is deduced from experimental values of the RBEs of ultrasoft X rays. The contribution to V79 cell inactivation solely due to the core ionizations in DNA is found to be 75 +/- 27% for energetic electrons and gamma rays. This surprisingly large contribution strongly suggests the presence of new mechanisms associated with critical lesions for cell inactivation.     

Cloning and mapping of the manganese superoxide dismutase gene (sodA) of Escherichia coli K-12.

An Escherichia coli gene bank composed of large DNA fragments (about 40 kilobases) was constructed by using the small cosmid pHC79. From it, a clone was isolated for its ability to overproduce superoxide dismutase. The enzyme overproduced was manganese superoxide dismutase, as determined by electrophoresis and antibody precipitation. Maxicell analysis and two-dimensional O'Farrell polyacrylamide gel electrophoresis demonstrated that the structural gene, sodA, of manganese superoxide dismutase was cloned. Subcloning fragments from the original cosmid located the sodA gene within a 4.8-kilobase EcoRI-BamHI fragment. This fragment was inserted into a lambda phage which was deleted for the att region and consequently could only lysogenize by recombination between the cloned bacterial DNA insertion and the bacterial chromosome. Genetic mapping of the prophage in such lysogens indicated that the chromosomal sodA locus lies near 87 min on the E. coli map.     

Some factors affecting successful vitrification of mouse blastocysts

The effects of temperature and exposure time to vitrification solutions on In vitro survival of mouse blastocysts were investigated. Blastocysts were first exposed for 10 min to vitrification Solution 1 (VS1) containing 10% glycerol-20% 1,2 propanediol in phosphate buffered saline (PBS), then to vitrification Solution 2 (VS2) with 25 % glycerol-25% 1,2 propanediol for various periods either at room temperature or at 4°C. At room temperature survival dropped quickly, while at 4°C an increase in survival was observed.

It is concluded that the viability of mouse blastocyts after vitrification is dependent on the temperature and duration of equilibration in vitrification solutions.     

Cloning and characterization of the pH 2.5 acid phosphatase gene, appA: Cyclic AMP mediated negative regulation

Summary The structural gene for an acid phosphatase coded for by the gene appA of Escherichia coli K12 was cloned from a cosmid library into pBR322 and the restriction map determined. Several appA deletion plasmids and a smaller appA ⁺ plasmid were constructed by in vitro recombination techniques and tested for their ability to complement an appA1 mutation. The appA gene was localized within a 2.1 kb segment. Its orientation was determined by construction of a hybrid plasmid carrying an appA-lacZ fusion. -galactosidase synthesized from the appA promoter was negatively regulated by cyclic AMP.     

Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase.

The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity. Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants. We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions. fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis. Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity. Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection. Functional recombinational repair was necessary for protection, but SOS induction was not involved. Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants. Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions. Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN. Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity. Furthermore, overexpression of SOD partially protected fur recA mutants. We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope.     

Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli: Fur (ferric uptake regulation) and Arc (aerobic respiration control)

The expression of sodA, the Escherichia coli gene encoding manganese superoxide dismutase (MnSOD) is induced by aerobiosis and superoxide generators such as paraquat. Analysis of variants expressing sodA in the absence of oxygen has revealed that mutations in genes for two global regulatory systems, Fur (ferric uptake regulation) and Arc (aerobic respiration control), are simultaneously required for the expression of sodA in anaerobiosis. The Fur protein still represses sodA in an iron-dependent fashion in aerobiosis. Moreover, all mutants remain inducible by paraquat, indicating that the positive control of SoxR, which mediates the response to superoxide in E. coli, is still operative. Thus, in addition to the response to the superoxide-mediated oxidative stress which depends on SoxR, two global controls regulate MnSOD expression: ArcA couples MnSOD expression to respiration, and Fur couples it to the intracellular concentration of iron.     

New 2-arylnaphthalenediols and triol inhibitors of HIV-1 integrase—Discovery of a new polyhydroxylated antiviral agent

A series of 13 hydroxylated 2-arylnaphthalenes have been synthesized and evaluated as HIV-1 integrase inhibitors. 7-(3,4,5-Trihydroxyphenyl)naphthalene-1,2,3-triol 1c revealed chemical instability upon storage, leading to the isolation of a dimer 5c which was also tested. In the 2-arylnaphthalene series, all compounds were active against HIV-1 IN with IC₅₀’s within the 1–10 μM range, except for 1c and 5c which displayed submicromolar activity. Antiviral activity against HIV-1 replication was measured on 1b–c and 5c. Amongst the tested molecules, only 5c was found to present antiviral properties with a low cytotoxicity on two different cell lines.