Macromolecular crowding accelerates the cohesion of DNA fragments with complementary termini.

Macromolecular crowding increases the rate of nonenzymatic cohesion of the complementary ends of lambda DNA. Both lambda DNA and DNA fragments bearing the cohesive ends of lambda DNA are similarly affected. High concentrations of plasma albumin or Ficoll 70 increase the rate of cohesion by ca. 100-fold whereas high concentrations of polyethylene glycol 8000 cause greater than 2000-fold stimulation in this rate. These results have implications for the mechanism of polymer-stimulated enzymatic ligation of DNA or RNA. In addition, these crowding effects may help to explain the rapid cohesion of lambda DNA observed in vivo. An improved procedure for the recovery of DNA fragments separated by agarose gel electrophoresis is also described.     

A monoclonal antibody to vasopressin: preparation, characterization, and application in immunocytochemistry

The hypothalamo-neurohypophysial system, containing the hormones oxytocin (OT) and vasopressin (VP) and their associated carrier proteins, the neurophysins (NPS), has been the subject of extensive investigation for more than 40 years. This system has been reinvestigated during the last decade by application of immunocytochemical methods employing the rabbit antisera to the hormones and NPS. In this study we describe the preparation and characterization of a monoclonal antibody to VP and its application in immunohistochemistry. The antibody did not cross-react with OT or arginine vasotocin (AVT). Its antigenic determinants as characterized by absorption with various VP analogs included two aromatic amino acids: Phe in position 3, and to a lesser extent Tyr in 2. Tissue fixation with formaldehyde resulted in inadequate immunostaining as compared to glutaraldehyde, most likely due to interference with the aromatic amino acid determinants by the former fixative.     

Optimal foraging: A case for random movement

Summary The bumblebee, Bombus flavifrons, forages randomly with respect to direction on Polemonium foliosissimum. This foraging pattern is as predicted for a system where there is a low probability of revisiting any given flower upon returning to a patch. This low revisitation probability is a function of the floral resource arrangement. It is further shown that B. flavifrons is using the resource distribution to direct its movements. A large percentage of all movements are to nearest neighbors with maximal foraging efficiency gained through minimization of flight distances.     

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.     

Charge neutralization and DNA bending by the Escherichia coli catabolite activator protein

We are interested in the role of asymmetric phosphate neutralization in DNA bending induced by proteins. We describe an experimental estimate of the actual electrostatic contribution of asymmetric phosphate neutralization to the bending of DNA by the Escherichia coli catabolite activator protein (CAP), a prototypical DNA-bending protein. Following assignment of putative electrostatic interactions between CAP and DNA phosphates based on X-ray crystal structures, appropriate phosphates in the CAP half-site DNA were chemically neutralized by methylphosphonate substitution. DNA shape was then evaluated using a semi-synthetic DNA electrophoretic phasing assay. Our results confirm that the unmodified CAP DNA half-site sequence is intrinsically curved by 26° in the direction enhanced in the complex with protein. In the absence of protein, neutralization of five appropriate phosphates increases DNA curvature to 32° (~23% increase), in the predicted direction. Shifting the placement of the neutralized phosphates changes the DNA shape, suggesting that sequence-directed DNA curvature can be modified by the asymmetry of phosphate neutralization. We suggest that asymmetric phosphate neutralization contributes favorably to DNA bending by CAP, but cannot account for the full DNA deformation.     

A ring-like nucleoid is not necessary for radioresistance in the <Emphasis Type="Italic">Deinococcaceae</Emphasis>

Background  

Transmission electron microscopy images of Deinococcus radiodurans R1 suggest that the nucleoid of this species exists as a "ring-like" body, and have led to speculation that this structure contributes to the radioresistance of the species. Since extreme radioresistance is characteristic of six other species of Deinococcus, we have attempted to correlate nucleoid morphology and radioresistance by determining whether the genomic DNA of each of these species exhibit similar structures.     

Introduction of the carrot HSP17.7 into potato (Solanum tuberosum L.) enhances cellular membrane stability and tuberization in vitro

We have examined the ability of a carrot (Daucus carota L.) heat shock protein gene encoding HSP17.7 (DcHSP17.7) to confer enhanced heat tolerance to potato (Solanum tuberosum L.), a cool-season crop. The DcHSP17.7 gene was fused to a 6XHistidine (His) tag to distinguish the engineered protein from endogenous potato proteins and was introduced into the potato cultivar 'Désirée' under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Western analysis showed that engineered DcHSP17.7 was constitutively, but not abundantly, expressed in transgenic potato lines before heat stress. Leaves from multiple regenerated potato lines that contain the transgene exhibited significantly improved cellular membrane stability at high temperatures, compared with wild-type and vector control plants. Transgenic potato lines also exhibited enhanced tuberization in vitro: under a condition of constant heat stress, at 29 degrees C, nodal sections of the transgenic lines produced larger and heavier microtubers at higher rates, compared to the wild type and vector controls. The dry weight and percentages of microtubers that were longer than 5 mm were up to three times higher in the transgenic lines. Our results suggest that constitutive expression of carrot HSP17.7 can enhance thermotolerance in transgenic potato plants. To our knowledge, this is the first study that shows that the thermotolerance of potato can be enhanced through gene transfer.     

Effects of MagPro™ on muscle performance

Athletes are on an endless quest to enhance performance and are frequently barraged by products that purport to contribute to various components of athletic activity. The purpose of this study was to determine if MagPro? influenced muscle flexibility or muscle endurance. This was a double-blind, randomized, controlled study using a repeated-measures design. The Institutional Review Board approved consent was obtained. The participants were healthy, physically active adults (n = 38 for phase 1; n = 18 for phase 2). Two creams were used: MagPro? (Mg cream) and a placebo. In phase 1, each cream was applied to the gastroc-soleus muscles. A stretching protocol was completed, and ankle dorsiflexion was compared. In phase 2, 1 cream was applied to both quadriceps muscles. An endurance protocol using a Life Fitness bicycle was completed. The procedure was repeated with the other cream on the quadriceps muscle 1 week later. For the flexibility phase, an analysis of variance with repeated measures revealed no difference between the 2 creams (p = 0.50), but there was a change in the flexibility over time (p = 0.00). For the endurance phase, paired t-tests revealed that there was no significant difference between the first (p = 0.26) or second (p = 0.35) cycling bouts of either cream. Likewise, there were no differences between the first and second cycling bouts of both the creams (MagPro? p = 0.46; Placebo p = 0.08). Despite previous studies demonstrating improved performance with Mg supplements, MagPro? did not enhance the outcome measures of this study. Examination of alternative application techniques and other outcome measures would be appropriate.     

A simple interferon assay as an adjunct for determining the genus of origin of cell cultures

Summary A short, simple test involving interferon-mediated protection of cells in vitro from cytopathogenic effects produced by vesicular stomatitis virus or Sindbis virus has been developed to help in determining the genus of origin of cells. By using the observed pattern of protection of five cell types by five interferons, cells could be grouped as of primate, rabbit, rat, and mouse origins. The primate grouping resulted from bilateral cross-reactions between the human and monkey systems. An unexpected observation was that African green monkey interferon preparations protect both monkey and rat cells, but not the converse. Chromosomal analysis of the cell cultures confirmed the genus determined by the interferon test.