Modified peptide nucleic acids are internalized in mouse macrophages RAW 264.7 and inhibit inducible nitric oxide synthase.

Overexpression of inducible nitric oxide synthase causes the production of high levels of nitric oxide, which, under pathological conditions, leads to immunosuppression and tissue damage. The results recently obtained using peptide nucleic acids, rather than traditional oligonucleotides as antigen and antisense molecules, prompted us to test their efficacy in the regulation of nitric oxide production, thereby overcoming the obstacle of cellular internalization. The cellular permeability of four inducible nitric oxide synthase antisense peptide nucleic acids of different lengths was evaluated. These peptide nucleic acids were covalently linked to a hydrophobic peptide moiety to increase internalization and to a tyrosine to allow selective 125I radiolabelling. Internalization experiments showed a 3-25-fold increase in the membrane permeability of the modified peptide nucleic acids with respect to controls. Inducible nitric oxide synthase inhibition experiments on intact stimulated macrophages RAW 264.7 after passive permeation of the two antisense peptide nucleic acids 3 and 4 demonstrated a significant decrease (43-44%) in protein enzymatic activity with respect to the controls. These data offer a basis for developing a good alternative to conventional drugs directed against inducible nitric oxide synthase overexpression.     

PCO(2) threshold for CNS oxygen toxicity in rats in the low range of hyperbaric PO(2).

Central nervous system (CNS) oxygen toxicity, as manifested by the first electrical discharge (FED) in the electroencephalogram, can occur as convulsions and loss of consciousness. CO(2) potentiates this risk by vasodilation and pH reduction. We suggest that CO(2) can produce CNS oxygen toxicity at a PO(2) that does not on its own ultimately cause FED. We searched for the CO(2) threshold that will result in the appearance of FED at a PO(2) between 507 and 253 kPa. Rats were exposed to a PO(2) and an inspired PCO(2) in 1-kPa steps to define the threshold for FED. The results confirmed our assumption that each rat has its own PCO(2) threshold, any PCO(2) above which will cause FED but below which no FED will occur. As PO(2) decreased from 507 to 456, 405, and 355 kPa, the percentage of rats that exhibited FED without the addition of CO(2) (F(0)) dropped from 91 to 62, to 8 and 0%, respectively. The percentage of rats (F) having FED as a function of PCO(2) was sigmoid in shape and displaced toward high PCO(2) with the reduction in PO(2). The following formula is suggested to express risk as a function of PCO(2) and PO(2) [abstract: see text] where P(50) is the PCO(2)for the half response and N is power. A small increase in PCO(2) at a PO(2) that does not cause CNS oxygen toxicity may shift an entire population into the risk zone. Closed-circuit divers who are CO(2)retainers or divers who have elevated inspired CO(2)are at increased risk of CNS oxygen toxicity.     

The influence of cell surface properties of thermophilic streptococci on attachment to stainlesssteel

The quality of milk products is threatened by the formation of biofilms of thermophilicstreptococci on the internal surfaces of plate heat exchangers used in milk processing. Althoughattachment to stainless steel surfaces is one of the first stages in the development of a biofilm, themechanisms involved in attachment have not been reported. The cell surface properties of 12strains of thermophilic streptococci were examined to determine their importance in attachment tostainless steel surfaces. Hydrophobicity, extracellular polysaccharide production and cell surfacecharge varied between the different strains but could not be related to numbers attaching. Treatingthe cells with sodium metaperiodate, lysozyme or trichloroacetic acid to disrupt cell surfacepolysaccharide had no effect on attachment. Treatment with trypsin or sodium dodecyl sulphate toremove cell surface proteins resulted in a 100-fold reduction in the number of bacteria attaching.This result suggests that the surface proteins of the thermophilic streptococci are important intheir attachment to stainless steel.     

Cloning and expression of the 3' portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage lambda gt11 T4 dC DNA library was screened for phage which produced a beta-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25% of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within lambda gt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native beta-galactosidase.     

Photosynthetic Development of Micropropagated Strawberry Plantlets following Transplanting

Leaves from plantlets of Fragaria x ananassa Duch. cv. Gentocultured in vitro do not fix sufficient carbon to maintain theplantlets without an added carbon source. Following transplantingto glasshouse conditions these leaves fail to develop significantphotosynthetic ability and degenerate. Those leaves developingsubsequent to transplanting are photosynthetically capable.The possible transitory, nutritive function of leaves producedin vitro is discussed. Fragaria x ananassa Duch., strawberry, in vitro culture, transplanting, photosynthesis     

Is prostaglandin E the neural mediator of the febrile response? The case against a proven obligatory role.

We have reviewed the evidence in favor of a prostaglandin mediator of the thermal responses in fever and found that PGE injected into the hypothalamus does not always cause fever, that cerebrospinal fluid concentrations of PGE are not reliable reflections of hypothalamic events, and that antipyretic drugs may act in ways other than inhibiting PGE synthesis. Fever is not blocked by prostaglandin antagonists, nor by ablation of PGE-sensitive areas of the brain. There is poor correlation between the effects of pyrogens and of PGE on cerebral neurons. There is evidence that at least one prostanoid other than prostaglandin is a mediator of fever, but the prostanoid has not been identified yet. We conclude that PGE may contribute to the neural responses in fever but is not essential.     

Mapping and analysis of protein sulfhydryl groups by modification with 2-bromoacetamido-4-nitrophenol

A method for identifying cysteine-containing peptides in proteins is presented using 2-bromoacetamido-4-nitrophenol (BNP) to introduce an easily detectable probe. The formation of a covalent bond between the protein sulfhydryl group and the acetamido moiety of BNP introduces a chromophore with an absorbance maximum at 410 nm. The modified protein can then be cleaved with appropriate proteases and the resulting peptides separated by chromatographic methods. Monitoring the effluent at a single wavelength (405 nm) provides a rapid and simple method of detecting and isolating only those peptides which contain cysteine residue(s). The nitrophenol derivative is stable under conditions required for protease cleavage. The reagent is therefore useful for locating cysteine-containing peptides in protein digests and can be used to explore the accessibility of different cysteines under a variety of conditions. The ease of modification, specificity of reaction, product stability, and simple detection of modified peptides make BNP ideal for investigation of cysteine residues.     

Southern analysis of BT-R 1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210?kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp. berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210?kDa protein, termed BT-R₁ (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R ₁ , the gene encoding the 210?kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R₁ as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R₁. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R₁, whereas the other is a closely related homologue.