The effect of cross-links on the conformational dynamics of duplex DNA.

The base pair lifetimes and apparent dissociation constants of a 21 base DNA hairpin and an analog possessing a disulfide cross-link bridging the 3'- and 5'-terminal bases were determined by measuring imino proton exchange rates as a function of exchange catalyst concentration and temperature. A comparison of the lifetimes and apparent dissociation constants for corresponding base pairs of the two hairpins indicates that the cross-link neither increases the number of base pairs involved in fraying nor alters the lifetime, dissociation constant, or the opened structure from which exchange occurs for the base pairs that are not frayed. The cross-link does, however, stabilize the frayed penultimate base pair of the stem duplex. Significantly, it appears that the disulfide cross-link is more effective at preventing fraying of the penultimate base pair than is the 5 base hairpin loop. Because this disulfide cross-link can be incorporated site specifically, and does not adversely affect static or dynamic properties of DNA, it should prove very useful in studies of nucleic acid structure and function.     

Genetic manipulation of plant growth-promoting bacteria to enhance biocontrol of phytopathogens

Plant growth-promoting bacteria (PGPB) control the damage to plants from phytopathogens by a number of different mechanisms including: outcompeting the phytopathogen, physical displacement of the phytopathogen, secretion of siderophores to prevent pathogens in the immediate vicinity from proliferating, synthesis of antibiotics, synthesis of a variety of small molecules that can inhibit phytopathogen growth, production of enzymes that inhibit the phytopathogen and stimulation of the systemic resistance of the plant. Biocontrol PGPB may be improved by genetically engineering them to overexpress one or more of these traits so that strains with several different anti-phytopathogen traits which can act synergistically are created. In engineering these strains it is essential to ensure that the normal functioning of the bacterium is not impaired, i.e., that there is no problem with metabolic load.     

Lateral hypothalamic lesions and striatal dopamine levels

Rats with bilateral lateral hypothalamic lesions were killed on the third day after surgery and their brains were assayed for tel-diencephalic norepinephrine and striatal dopamine. Lesion-induced weight loss was highly correlated with depletion of striatal dopamine but not with tel-diencephalic norepinephrine. In rats with severe dopamine depletions, the degree of weight loss was related more to the striatum with the highest remaining level of dopamine suggesting that a critical level of dopamine in one striatum may be essential for lateral hypothalamic recovery.     

The antitumor agent 3-bromopyruvate has a short half-life at physiological conditions

Clinical research is currently exploring the validity of the anti-tumor candidate 3-bromopyruvate (3-BP) as a novel treatment for several types of cancer. However, recent publications have overlooked rarely-cited earlier work about the instability of 3-BP and its decay to 3-hydroxypyruvate (3-HP) which have obvious implications for its mechanism of action against tumors, how it is administered, and for precautions when preparing solutions of 3-BP. This study found the first-order decay rate of 3-BP at physiological temperature and pH has a half-life of only 77 min. Lower buffer pH decreases the decay rate, while choice of buffer and concentration do not affect it. A method for preparing more stable solutions is also reported.     

Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.     

1-Aminocyclopropane-1-carboxylate deaminase from Pseudomonas putida UW4 facilitates the growth of canola in the presence of salt

The growth of canola plants treated with either wild-type Pseudomonas putida UW4 or a 1-aminocyclopropane-1-carboxylate (ACC) deaminase minus mutant of this strain was monitored in the presence of inhibitory levels of salt, i.e., 1.0 mol/L at 10 degrees C and 150 mmol/L at 20 degrees C. This strain is psychrotolerant with a maximal growth rate of approximately 30 degrees C and the ability to proliferate at 4 degrees C. Although plant growth was inhibited dramatically by the addition of 1.0 mol/L salt at 10 degrees C and only slightly by 150 mmol/L salt at 20 degrees C under both sets of conditions, the addition of the wild type but not the mutant strain of P. putida UW4 significantly improved plant growth. This result confirms the previous suggestion that bacterial strains that contain ACC deaminase confer salt tolerance to plants by lowering salt-induced ethylene synthesis.     

Cdc1p is an endoplasmic reticulum-localized putative lipid phosphatase that affects Golgi inheritance and actin polarization by activating Ca2+ signaling

In the budding yeast Saccharomyces cerevisiae, mutations in the essential gene CDC1 cause defects in Golgi inheritance and actin polarization. However, the biochemical function of Cdc1p is unknown. Previous work showed that cdc1 mutants accumulate intracellular Ca(2+) and display enhanced sensitivity to the extracellular Mn(2+) concentration, suggesting that Cdc1p might regulate divalent cation homeostasis. By contrast, our data indicate that Cdc1p is a Mn(2+)-dependent protein that can affect Ca(2+) levels. We identified a cdc1 allele that activates Ca(2+) signaling but does not show enhanced sensitivity to the Mn(2+) concentration. Furthermore, our studies show that Cdc1p is an endoplasmic reticulum-localized transmembrane protein with a putative phosphoesterase domain facing the lumen. cdc1 mutant cells accumulate an unidentified phospholipid, suggesting that Cdc1p may be a lipid phosphatase. Previous work showed that deletion of the plasma membrane Ca(2+) channel Cch1p partially suppressed the cdc1 growth phenotype, and we find that deletion of Cch1p also suppresses the Golgi inheritance and actin polarization phenotypes. The combined data fit a model in which the cdc1 mutant phenotypes result from accumulation of a phosphorylated lipid that activates Ca(2+) signaling.