Changes in RNA polymerase II in a cell cycle-specific temperature-sensitive mutant of hamster cells

tsAF8 cells are a temperature-sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G₁ phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bind [³H]-γ-amanitin decrease in tsAF8 cells at 40.6°, with a half-life of ～ 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34° or in BHK cells at either 34° or 40.6°. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6°. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage.     

Marker rescue transformation by linear plasmid DNA in Bacillus subtilis

Although plasmid-free Bacillus subtilis cannot be transformed for markers carried by linear or nicked plasmid DNA, a resident plasmid can rescue a marker on such damaged DNA under certain conditions. Linearized chimeric plasmid DNA has been used to transform cultures carrying a resident plasmid which is homologous with a portion of the donor. This system has revealed the following properties of the marker rescue process: (1) It is recE dependent. (2) It requires the presence in the resident plasmid of sequences which are homologous to the donor. (3) When the selected marker is on a nonhomologous segment it must be flanked by segments which are homologous to the resident plasmid. (4) The efficiency of rescue varies in a regular way with the position of the linearizing cut. (5) Marker rescue is first order with respect to DNA concentration. These properties and other data are interpreted as providing a strong indication that marker rescue occurs by recombination, although an alternative explanation involving recE-dependent recircularization of the donor plasmid has not been eliminated. Our results also suggest that if the major pathway of marker rescue is by recombination, an average of 0.15 Mdal (single strand) must be removed from each donor DNA molecule or otherwise rendered unavailable for recombination and that the exchange frequency during transformational recombination is approximately 0.2 to 0.5 Mdal⁻¹.     

Interactions of cardiac glycosides with cells and membranes Properties and structural aspects of two receptor sites for ouabain in erythrocytes

The existence of two types of binding sites for ouabain in human erythrocyte membranes is described. Receptor sites designated as ‘type I’, which may be identical to the K⁺-insensitive sites of intact cells, were detected at concentrations of ouabain as low as 10⁻⁷ M. The ‘type II’ receptor sites require the inclusion of Mg²⁺ + P_i to form complexes with ouabain; they may be identical to the K⁺-sensitive sites of intact cells. These sites were saturated at approx. 5 · 10⁻⁷ M ouabain but could not be detected at higher concentrations. The range of ouabain concentrations at which ‘type I’ receptors start to predominate (i.e. 5 · 10⁻⁸–5 · 10⁻⁷ M) was termed ‘critical digitalis concentrations’. The process of binding reached equilibrium within 1 and 4 h for ‘type I’ and ‘type II’ sites, respectively. The dissociation constant for ‘type II’ receptor-ouabain complexes was 7.6 · 10⁻⁹ M.Under similar experimental conditions, rat erythrocyte membranes exhibited only non-saturable sites.Alterations in the proportions of the two types of receptors were demonstrated by preincubation of the membranes, in the presence or absence of Mg²⁺ + P_i, prior to the addition of ouabain. In the first case, ‘type II receptor-ouabain’ complexes were stabilized at about 50% of the untreated membranes and ‘type I-ouabain’ complexes slowly approached equilibrium over a period of 24 h. In the latter instance, ‘type I’ receptors were not detected, and only ‘type II-ouabain’ complexes prevailed.     

Periovulatory changes in ovarian prostaglandin formation and their hormonal control in the rat

The concentration of prostaglandins of the E-group (PGE) and F-group (PGF) and the activity of prostaglandin-synthetase in rat ovaries increased on the evening of the day of proestrus and reached a peak at 5.00 h on the following morning, i.e. about the time of ovulation. Enzyme activity and PG concentrations receded to basal levels by 10.00 h on the day of estrus. These changes were prevented when the proestrous gonadotropin surge was blocked by administration of nembutal, and could be restored by administration of either LH or of FSH freed of LH contamination. The spontaneous preovulatory rise in prostaglandin concentration was about 6-fold for PGF and 30-fold for PGE, compared with values observed during the remainder of the cycle, whereas the rise in prostaglandin synthetase activity was only about 1.7-fold. The LH effect on PG accumulation had a latency of 2–4 h, which argues for enzyme synthesis rather than activation of preformed enzyme as the mechanism responsible. The small magnitude of the change in enzymic activity suggests that LH may, in addition, augment the availability of PG precursors. The results are compatible with the concept that prostaglandins play a physiological role in the gonadotropin-induced process of follicular rupture.     

Different Placebos,Different Mechanisms,Different Outcomes: Lessons for Clinical Trials

Clinical trials use placebos with the assumption that they are inert, thus all placebos are considered to be equal. Here we show that this assumption is wrong and that different placebo procedures are associated to different therapeutic rituals which, in turn, trigger different mechanisms and produce different therapeutic outcomes. We studied high altitude, or hypobaric hypoxia, headache, in which two different placebos were administered. The first was placebo oxygen inhaled through a mask, whereas the second was placebo aspirin swallowed with a pill. Both placebos were given after a conditioning procedure, whereby either real oxygen or real aspirin was administered for three consecutive sessions to reduce headache pain. We found that after real oxygen conditioning, placebo oxygen induced pain relief along with a reduction in ventilation, blood alkalosis and salivary prostaglandin (PG)E2, yet without any increase in blood oxygen saturation (SO2). By contrast, after real aspirin conditioning, placebo aspirin induced pain relief through the inhibition of all the products of cyclooxygenase, that is, PGD2, PGE2, PGF2, PGI2, thromboxane (TX)A2, without affecting ventilation and blood alkalosis. Therefore, two different placebos, associated to two different therapeutic rituals, used two different pathways to reduce headache pain. The analgesic effect following placebo oxygen was superior to placebo aspirin. These findings show that different placebos may use different mechanisms to reduce high altitude headache, depending on the therapeutic ritual and the route of administration. In clinical trials, placebos and outcome measures should be selected very carefully in order not to incur in wrong interpretations.     

Host selection as a downstream strategy: polyelectrolyte precipitation of beta-glucuronidase from plant extracts.

Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors.     

Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.