A lung type prostaglandin F synthase is expressed in bovine liver: cDNA sequence and expression in E. coli.

Using the cDNA of bovine lung prostaglandin F synthase (EC 1.1.1.2) as a probe, we isolated a clone from a bovine liver cDNA library which differed in only eleven nucleotides from the probe. The corresponding protein contained three amino acid substitutions, including a leucine residue which is conserved throughout all aldo-keto reductases. We inserted the liver cDNA into expression vector pUC19 and expressed the recombinant liver enzyme in E.coli. The purified liver enzyme reduced prostaglandin H2 as well as prostaglandin D2 and various carbonyl compounds. The high relative activity against prostaglandin H2 in combination with a high Km value for prostaglandin D2 identified this liver enzyme as a lung type prostaglandin F synthase. However, the binding constant for NADPH of the liver enzyme was 3.5 fold higher than that of lung prostaglandin F synthase.     

Enzymic activity of salivary amylase when bound to the surface of oral streptococci

The enzymatic activity of salivary amylase bound to the surface of several species of oral streptococci was determined by the production of acid from starch and by the degradation of maltotetraose to glucose in a coupled, spectrophotometric assay. Most strains able to bind amylase exhibited functional enzyme on their surface and produced acid from the products of amylolytic degradation. These strains were unable to utilise starch in the absence of salivary amylase. Two strains failed to produce acid from starch, despite the presence of functional salivary amylase, because they could not utilise maltose. Strains that could not bind salivary amylase failed to produce acid from starch. In no case was all the bound salivary amylase active, and two strains of Streptococcus mitis which bound amylase did not exhibit any enzyme activity on their cell surface. The ability to bind amylase may confer a survival advantage on oral bacteria which inhabit hosts that consume diets containing starch.     

The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter

The influence of low temperature on the operation of the xanthophyll cycle and energy dissipation activity, as ascertained through measurements of chlorophyll fluorescence, was examined in two broad-leaved evergreen species, Vinca minor L. and Euonymus kiautschovicus Loessner. In leaves examined under laboratory conditions, energy dissipation activity developed more slowly at lower leaf temperatures, but the final, steady-state level of such activity was greater at lower temperatures where the rate of energy utilization (through photosynthetic electron transport) was much lower. The rate at which energy dissipation activity increased was similar to that of the de-epoxidation of violaxanthin to antheraxanthin and zea-xanthin at different temperatures. However, leaves in the field examined prior to sunrise on mornings following cold days and nights exhibited a retention of antheraxanthin and zeaxanthin that was associated with sustained decreases in photosystem II efficiency. We therefore suggest that this phenomenon of ‘photoinhibition’ in response to light and cold temperatures during the winter results from sustained photoprotective thermal energy dissipation associated with the xanthophyll cycle. Such retention of the de-epoxidized components of the xanthophyll cycle responded to day-to-day changes in temperature, being greatest on the coldest mornings (when photoprotective energy dissipation might be most required) and less on warmer mornings when photosynthesis could presumably proceed at higher rates.     

Vector competence of Culicoides bolitinos and C. imicola for South African bluetongue virus serotypes 1, 3 and 4

Abstract .The susceptibility of field-collected Culicoides bolitinos to infection by oral ingestion of bluetongue virus serotypes 1, 3 and 4 (BLU 1, 3 and 4) was compared with that of field-collected C. imicola and laboratory reared C. variipennis sonorensis . The concentration of the virus per millilitre of bloodmeal was 10^5.0 and 10^6.0TCID₅₀ for BLU 4 and 10^7.2TCID₅₀ for BLU 1 and 3. Of 4927 C. bolitinos and 9585 C. imicola fed, 386 and 287 individual midges survived 10 days extrinsic incubation, respectively. Midges were assayed for the presence of virus using a microtitration assay on BHK-21 cells and/or an antigen capture ELISA. Infection prevalences for the different serotypes as determined by virus isolation ranged from 22.7 to 82.0% in C. bolitinos and from 1.9 to 9.8% in C. imicola; infection prevalences were highest for BLU 1, and lowest for BLU 4 in both species. The mean log₁₀ TCID₅₀ titre of the three BLU viruses per single fly was higher in C. bolitinos than in C. imicola . The results suggested that C. bolitinos populations are capable vectors of the BLU viruses in South Africa. A high correlation was found between virus isolation and ELISA results for the detection of BLU 1, and less for BLU 4; the ELISA failed to detect the presence of BLU 3 in infected flies. The C. v. sonorensis colonies had a significantly lower susceptibility to infection with BLU 1, 3 and 4 than C. bolitinos and C. imicola . However, since infection prevalence of C. v. sonorensis was determined only by ELISA, this finding may merely reflect the insensitivity of this assay at low virus titres, compared to virus isolation.     

Purified Fc epsilon R+ bone marrow and splenic non-B, non-T cells are highly enriched in the capacity to produce IL-4 in response to immobilized IgE, IgG2a, or ionomycin.

Non-B, non-T cells from spleen and bone marrow cells produce IL-4 in response to cross-linkage of high affinity receptors for Fc epsilon R or Fc gamma RII, and to treatment with calcium ionophores. Cells bearing high affinity Fc epsilon R constituted 1 to 2% of non-B, non-T cells of spleen and of total bone marrow cells from naive donors. In mice whose immune systems had been polyclonally activated by injection with anti-IgD antibodies or had been infected with Nippostrongylus brasiliensis larvae, the frequency of Fc epsilon R+ cells in splenic non-B, non-T cells was also 1 to 2% but in bone marrow from anti-IgD-injected mice donors the frequency was approximately 5%. Cell sorting experiments revealed that all of the capacity to produce IL-4 in response to immobilized IgE or IgG2a or to ionomycin was found in the Fc epsilon R+ fraction. Among the Fc epsilon R+ spleen cells from naive donors, the frequency of IL-4-producing cells was 1/20 to 1/40 whereas in mice that had been injected with anti-IgD or infected with N. brasiliensis, the frequency of IL-4 producing cells in the Fc epsilon R+ population was approximately 1/5.     

Localization of deletion breakpoints in radiation-induced mutants of the hprt gene in hamster cells

DNA was analysed from a large set of hamster hprt gene mutants, some induced by ionising radiations and others occurring naturally, to identify those with large alterations in part of the gene. DNA from these mutants was restricted further with different endonucleases and probed to establish the patterns of restriction fragments remaining. Of 15 mutants characterized, one showed a duplication of part of the 5' end of the gene, and the remainder showed deletions of various sizes. It was possible to approximately locate the breakpoints of the deletions by comparison of fragment patterns to a recently-established map of the hamster gene. The relatively small number of mutants examined precludes rigorous analysis of the distribution of breakpoints in the hprt gene, but taken with other recent studies of deletion mutagenesis it is suggested that non-random induction or selection of this type of mutation may occur.     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.