Occurrence of plasmid DNA in the sepiolid squid symbiontVibrio fischeri

Because of the importance of plasmids in many bacterial associations with plants and animals, we determined the occurrence and distribution of plasmid DNA in symbioticVibrio fischeri from the light organ of the sepiolid squidEuprymna scolopes. Analyses of 225 isolates of symbioticV. fischeri from 25 individual squids revealed an overall plasmid-carriage rate of 56%. A large plasmid (39 kb) was detected in 96% of those isolates carrying plasmids, and multiple small plasmids were found to co-occur with one of the large plasmids in 81% of plasmid-carrying strains. In addition, these plasmids appear to be restricted toV. fischeri strains isolated fromE. scolopes and from seawater at sites of squid populations. We were unable to assign a role or function to these plasmids, but they do not carry genes required for the establishment of the light organ symbiosis. We conclude that the essential bacterial symbiotic determinants must be encoded on the chromosome and that the plasmids may carry genes that are important for the survival of theseV. fischeri strains outside of the symbiotic association.     

Simulation of Tumor-Specific Delivery of Radioligand Comparison of One Step,Two Step,and Genetic Transduction Systems

A mathematical model simulation was performed to estimate the amount of radioactivity in plasma, normal tissues, and tumor tissue through three delivery approaches: one step radiolabeled monoclonal antibody (MAb) CC49 i.v. bolus injection, two step method with biotin conjugated CC49 i.v. bolus injection followed 72 hours later by i.v. bolus radiolabeled streptavidin injection, and gene therapy method to express biotin on the tumor cell surface followed by i.v. bolus radiolabeled streptavidin injection. The mathematical model was built based on a system of ordinary differential equations consisting of inputs and outputs of model components in plasma, normal tissues, and tumor tissue. Through computer modeling, we calculated concentrations of each component for plasma, tumor and normal tissues at various time points. Radioactivity ratios of tumor to plasma and tumor to normal tissues increased with time. The increase of tumor to normal tissue ratios was much faster for the gene therapy approach than for single step and two step approaches, e.g., a ratio of 24.26 vs. 2.06 and 6.24 at 72 hours after radioligand injection. Radioactivity ratios predicted by the model varied with the amount of radioactivity injected and the time interval between injections. The model could be used to evaluate different radioimmunotherapy strategies and to predict radioactivity biodistribution using other receptor-ligand systems.     

Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector.

An integration vector for gene analysis in Streptomyces has been constructed. This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome. To overcome methylation-specific restriction barriers, an E. coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids. The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA. Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S. avermitilis DNA, were used in complementation analyses of seven S. avermitilis mutants defective in glycosylation of avermectin (Av). Three complementation groups, located in a 7-kb region, were identified. Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer. Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.     

HER-2/neu oncogene expression, DNA ploidy and proliferation index in breast cancer.

HER-2/neu gene expression, DNA ploidy and proliferation index were studied in 250 cases of breast cancer. Expression of HER-2/neu was determined by using an antibody to the HER-2/neu receptor. Ki-67 antibody was used to determine the proliferation index of the breast cancers, and the Feulgen method was used to assess DNA amounts in the tumor cells. Histochemical staining was quantitated by image analysis. Of the cancers studied, 72 were positive for overexpression of HER-2/neu protein; of these, 62 (86%) possessed near-tetraploid DNA content, and 47 (65%) had more than one G0G1 stem line (polyploid) of DNA distribution. Cells from the cases negative for HER/2-neu overexpression contained DNA amounts that ranged from diploid to varying degrees of aneuploid. A significant difference in the amounts of cellular proliferation in HER-2/neu overexpressing cancers was found between those that expressed the HER-2/neu receptor on their membranes and those that exhibited mainly cytoplasmic receptors.     

Three-dimensional knee joint loading during seated cycling.

The hypothesis which motivated the work reported in this article was that neglecting pure moments developed between the foot and pedal during cycling leads to a substantial error in computing axial and varus/valgus moments at the knee. To test this hypothesis, a mathematical procedure was developed for computing the three-dimensional knee loads using three-dimensional pedal forces and moments. In addition to data from a six-load-component pedal dynamometer, the model used pedal position and orientation and knee position in the frontal plane to determine the knee joint loads. Experimental data were collected from the right leg of 11 male subjects during steady-state cycling at 90 rpm and 225 W. The mean peak varus knee moment calculated was 15.3 N m and the mean peak valgus knee moment was 11.2 N m. Neglecting the pedal moment about the anterior/posterior axis resulted in an average absolute error of 2.6 N m and a maximum absolute error of 4.0 N m in the varus/valgus knee moment. The mean peak internal and external axial knee moments were 2.8 N m and 2.3 N m, respectively. The average and maximum absolute errors in the axial knee moment for not including the moment about an axis normal to the pedal were found to be 2.6 N m and 5.0 N m, respectively. The results strongly support the use of three-dimensional pedal loads in the computation of knee joint moments out of the sagittal plane.     

Reproducibility of Dual-Energy X-ray Absorptiometry Measurements in Prepubertal Girls

Objective : Regulation of growth and development, clinical assessment, and obesity are among the areas of nutritionrelated research, wherein accurate assessment of body composition is important. We want to test the hypothesis that dual-energy X-ray absorptiometry (DXA) measurements are reproducible in healthy girls. Research Methods and Procedures : We determined total body composition measurements in healthy prepubertal girls using DXA twice, 6 weeks apart. Results : We studied 61 healthy, normal-weight, prepubertal girls, aged 4.8 years to 10.3 years. The girls' DXA-derived mean weight between visits 1 and 2 significantly increased (27.14 kg vs. 27.80 kg, P<0.0001). The increased weight was due to significant increases in total body fat-free mass (FFM) (19.53 kg vs. 19.89 kg,P<O.001), total body bone mass (1.05 kg vs. 1.07 kg, P<0.0001), and total body fat mass (7.61 kg vs. 7.91 kg,P<0.03). The girls' DXA-derived mean total trunk mass between visits 1 and 2 significantly increased (11.23 kg vs. 11.63 kg, p<<0.0001), as did total leg mass (9.33 kg vs. 9.53 kg, p<<0.001), although no significant differences were observed in total arm mass (2.52 kg vs. 2.54 kg, p< = 0.37). The Pearson coefficient of correlation (r) and total coefficient of variation (CV) for intraindividual measurements by DXA were: weight—r = 0.99, CV= 1.97%; total body FFM—r = 0.96, CV = 2.30%; total body bone mass—r = 0.99, CV = 2.08%; total body fat mass—r = 0.96, CV = 6.55%; percentage total body fat— r = 0.91, CV = 5.69%; total trunk mass—r = 0.96, CV = 3.59%; total arm mass—r = 0.95, CV = 4.09%; and total leg mass—r = 0.99, CV = 2.75%. Discussion : Total body FFM, total body bone mass, total body fat mass, percentage of total body fat mass, as well as regional mass determinations by DXA, were highly reproducible in healthy, normal-weight, prepubertal girls. We highly recommend the use of DXA for total body composition studies in girls aged 5 years to 10 years.     

Pyruvate production and excretion by the luminous marine bacteria.

During aerobic growth on glucose, several species of luminous marine bacteria exhibited an imcomplete oxidative catabolism of substrate. Pyruvate, one of the products of glucose metabolism, was excreted into the medium during exponential growth and accounted for up to 50% of the substrate carbon metabolized. When glucose was depleted from the medium, the excreted pyruvate was promptly utilized, demonstrating that the cells are capable of pyruvate catabolism. Pyruvate excretion is not a general phenomenon of carbohydrate metabolism since it does not occur during the utilization of glycerol or maltose. When cells pregrown on glycerol were exposed to glucose, they began to excrete pyruvate, even if protein synthesis was blocked with chloramphenicol. Glucose thus appears to have an effect on the activity of preexisting catabolic enzymes.