No abstract

RecA protein promotes a limited DNA strand exchange reaction, without ATP hydrolysis, that typically results in formation of short (1-2 kilobase pairs) regions of hybrid DNA. This nascent hybrid DNA is extended in a reaction that can be coupled to ATP hydrolysis. When ATP is hydrolyzed, the extension phase is progressive and its rate is 380 ؎ 20 bp min ؊1 at 37°C. A single RecA nucleoprotein filament can participate in multiple DNA strand exchange reactions concurrently (involving duplex DNA fragments that are homologous to different segments of the DNA within a nucleoprotein filament), with no effect on the observed rate of ATP hydrolysis. The ATP hydrolytic and hybrid DNA extension activities exhibit a dependence on temperature between 25 and 45°C that is, within experimental error, identical. This provides new evidence that the two processes are coupled. Arrhenius activation energies derived from the work are 13.3 ؎ 1.1 kcal mole ؊1 for DNA strand exchange, and 14.4 ؎ 1.4 kcal mole ؊1 for ATP hydrolysis during strand exchange. The rate of branch movement in the extension phase (base pair min ؊1 ) is related to the k cat for ATP hydrolysis during strand exchange (min ؊1 ) by a factor equivalent to 18 bp throughout the temperature range examined. The 18-base pair factor conforms to a quantitative prediction derived from a model in which ATP hydrolysis is coupled to a facilitated rotation of the DNA substrates. RecA filaments possess an intrinsic capacity for DNA strand exchange, mediated by binding energy rather than ATP hydrolysis, that is augmented by an ATP-dependent molecular motor.

Most osteoporosis drugs act by inhibiting bone resorption. A need exists for osteoporosis therapies that stimulate new bone formation. 2-Methylene-19-nor-(20S)-1a,25-dihydroxyvitamin D 3 (2MD) is a vitamin D analogue that potently stimulates bone formation activity in vitro and in the ovariectomized rat model. In this randomized, double-blind, placebo-controlled study of osteopenic women, the effect of daily oral treatment with 2MD on bone mineral density (BMD), serum markers of bone turnover, and safety were assessed over 1 year. Volunteers were randomly assigned to three treatment groups: placebo (n ¼ 50), 220 ng of 2MD (n ¼ 54), and 440 ng of 2MD (n ¼ 53). In general, 2MD was well tolerated. Although 2MD caused a marked increase in markers of bone formation, it did not significantly increase BMD. Since 2MD also shows marked activity on bone resorption (as revealed by dose-dependent increases in serum C-telopeptide crosslinks of type I collagen in this study), 2MD likely stimulated both bone formation and bone resorption, thereby increasing bone remodeling. ß

A 2014 multistate listeriosis outbreak was linked to consumption of caramel-coated apples, an unexpected and previously unreported vehicle for Listeria monocytogenes. This outbreak was unanticipated because both the pH of apples (<4.0) and the water activity of the caramel coating (<0.80) are too low to support Listeria growth. In this study, Granny Smith apples were inoculated with approximately 4 log10 CFU of L. monocytogenes (a cocktail of serotype 4b strains associated with the outbreak) on each apple’s skin, stem, and calyx. Half of the apples had sticks inserted into the core, while the remaining apples were left intact. Apples were dipped into hot caramel and stored at either 7°C or 25°C for up to 11 or 28 days, respectively. Data revealed that apples with inserted sticks supported significantly more L. monocytogenes growth than apples without sticks under both storage conditions. Within 3 days at 25°C, L. monocytogenes populations increased >3 log10 in apples with sticks, whereas only a 1-log10 increase was observed even after 1 week for caramel-coated apples without sticks. When stored at 7°C, apples with sticks exhibited an approximately 1.5-log10 increase in L. monocytogenes levels at 28 days, whereas no growth was observed in apples without sticks. We infer that insertion of a stick into the apple accelerates the transfer of juice from the interior of the apple to its surface, creating a microenvironment at the apple-caramel interface where L. monocytogenes can rapidly grow to levels sufficient to cause disease when stored at room temperature.

We present evidence for methyl ( Numerous biochemical processes are regulated by posttranslational modification of proteins. In bacterial chemotaxis the reversible methylation of certain membrane-bound receptor proteins appears to play an important role in the adaptation of bacteria to chemical stimuli (8)(9)(10)15). The specific functions these methyl-accepting chemotaxis proteins (MCPs) have in bacterial chemotaxis are not well understood, although changing levels of methylation of these proteins have been associated with the adaptation of bacteria to various attractants in Escherichia coli (5,9,13,15 described previously (14, 17). S. aureus V8 protease solution is 0.5 ,ug of protease per ml in a buffer of 40 mM Tris hydrochloride, 0.2% sodium dodecyl sulfate (SDS), 2 mM EDTA, and 40% glycerol (pH 7.8) (4).In vivo methylations. In vivo methylations were performed as described by Ullah and Ordal (17). Washed cells were suspended in protoplast buffer with 1 mg of lysozyme per ml. Methylation was initiated by the addition of [3H]methionine. In pulse-chase experiments the period of methylation was followed by the addition of a 100-fold excess of nonradioactive methionine for 2 min. Effector solutions were then added in some experiments for 30 s, at which time the reaction was terminated by freezing in a dry ice-acetone bath. The suspensions were thawed, and the protoplasts were pelleted at 4°C.SDS-PAGE. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli (11). After the addition of 100 ,ul of Laemmli sample buffer, the samples were boiled for 7 min and fractionated by SDS