Clostridium perfringens-Escherichia coli Shuttle Vectors That Carry Single Antibiotic Resistance Determinants

Two versatile Clostridium perfringens-Escherichia coli shuttle vectors were constructed. Each plasmid carried a single antibiotic resistance gene which was expressed in both organisms. The plasmid pJIR750 encoded resistance to chloramphenicol and pJIR751 encoded resistance to erythromycin. Each plasmid contained the pUC18-derived multiple cloning site and the lacZ′ gene which enabled direct screening for recombinants in E. coli . These plasmids should prove invaluable for the genetic manipulation of C. perfringens.     

Benthic community modification and recovery following intensive grazing byStrongylocentrotus droebachiensis

IntensiveStrongylocentrotus droebachiensis grazing of a previously sampled one-hectare plot provided an opportunity for quantitatively investigating grazing impact on the benthic macrophyte community and community recovery. The changes induced by grazing are compared with changes in a similar plot located nearby which was not grazed. A total of 154 quantitative 0.25 m² quadrats were collected in mid-summer, from the two sites, over a 4-year period. The results presented indicate thatS. droebachiensis populations in the Strait of Georgia undergo periodic, environmentally controlled, outbreaks and that these outbreaks are responsible for localized perturbation of the benthic macrophyte community. Short-term grazing, where the urchins do not remain in the area, result in a 60 percent reduction in most community parameters measured. The community requires 2 or 3 years to regain or exceed pre-grazing biomass levels while other parameters had not recovered after 3 years. An estimated 4–6 year period is required to achieve ecological climax (Fishelson, 1977) and species enrichment follows. The largest macrophyte in the Strait of Georgia, the kelpNereocystis luetkeana, is a successional species whose abundance is controlled, in part, by the successional status of the community.     

Cell preservation in a programmed cooling machine: the effect of variations in supercooling

When cells are cryopreserved in programmed cooling machines, they supercool to a variable and uncontrolled extent. Experiments were carried out with three cell-types (human peripheral lymphocytes, Chinese hamster lung fibroblasts, and mouse lymphoma cells) to determine whether there was any effect of supercooling on cell survival. Samples were cooled at 1 °C min^?1 in the presence of 12% v/v dimethyl sulphoxide (Me₂SO) to ?100 °C, and then thawed rapidly in a 37 °C water bath. There was no correlation between the extent of supercooling or the maximum cooling rate after freezing and cell survival, but the time taken for the sample temperature to return to the temperature at which freezing occurred did influence the survival of the two tissue culture cell lines. These results are interpreted on the basis of current theories according to which cells require sufficient time to lose water as they cool in order to avoid subsquent intracellular freezing, but must be cooled sufficiently rapidly to minimise solution effects. It is concluded that the variations in supercooling that occur in programmed cooling machines present no particular difficulties, providing appropriate cooling rates are chosen.     

The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.     

Total and regional cerebral blood flow during moderate and severe exercise in miniature swine.

To determine the influence of exercise on cerebral blood flow, we ran 14 swine at 3-6 mph and at 0-10% grades on a treadmill for 30 min at moderate and severe levels of exercise. Measuring heart rate, cardiac output, and aortic pressure via implanted probes, we injected 15-mum radiolabeled microspheres via the left atrium before and during exercise. We measured their distribution by gamma spectrometry, determining total cerebral blood flow, regional blood flow, and ratio of flow to gray and white matter. Heart rate, cardiac output, and aortic pressure rose progressively with increasing exercise. Total cerebral flow resembled that reported in humans, i.e., it did not change significantly with exercise. Regional flow distribution also failed to change significantly with exercise. The ratio of gray to white matter flow did not change except to the cerebellum where it rose significantly from resting values at both moderate and severe exercise. Gray matter received more flow than white matter during all three conditions of observation. Cerebral blood flow was remarkably constant during even severe exercise.     

Design and synthesis of indolo[2,3-a]quinolizin-7-one inhibitors of the ZipA-FtsZ interaction

The binding of FtsZ to ZipA is a potential target for antibacterial therapy. Based on a small molecule inhibitor of the ZipA-FtsZ interaction, a parallel synthesis of small molecules was initiated which targeted a key region of ZipA involved in FtsZ binding. The X-ray crystal structure of one of these molecules complexed with ZipA was solved. The structure revealed an unexpected binding mode, facilitated by desolvation of a loosely bound surface water.     

A modification of the Roe procedure for determination of fructose in tissues with increased specificity

A new modification of the procedure of Roe for determination of fructose concentrations in tissues is described. The modification shows no reaction with glucose or glucose phosphates. Tissues are homogenized in dilute perchloric acid, filtered, and reacted with alcoholic resorcinol and 30% HCl for 1 hr at 80°C. As little as 0.33 μg/ml of fructose may be reliably determined. Fructose phosphates react in this test but to a lesser extent than pure fructose.