Standardization of the chromium-51 release, cell-mediated cytotoxicity assay: cryopreservation of mouse effector and target cells

Utilizing controlled cryopreservation techniques, we were able to standardize the 51Cr release cytotoxicity assay and thereby ensured reliable comparisons between results obtained on different days. Optimal conditions for freezing of both effector and target cells were quite similar. Dimethyl sulfoxide (DMSO) at a concentration of 7.5-10.0% was employed as the cryoprotective agent and cells were frozen at the rate of -1 degrees C/minute. The handling procedures for the cells before and after freezing were important. Factors affecting recovery of functional reactivity were related to toxicity of DMSO for the cells, the osmotic stress placed upon the cells as the DMSO was being removed after thawing, the handling temperature of the freshly thawed cells, and the susceptibility of cells to mechanical damage immediately after thawing. The recovery of lymphocytes after freezing was about 70%; the recovery of cytotoxicity was around 85%. Syngeneic cytotoxic reactivity induced by inoculation with the Moloney strain of murine sarcoma virus was cryopreserved, as were allogeneic cytotoxicity and natural cytotoxic reactivity. Multiple tests employing effector cells from the same frozen pool gave reproducible results; the standard error of the mean percent cytotoxicity was less than 1.5%. Cryopreserved target cells gave decreased day-to-day variability in susceptibility to lysis, since the same population of cells could be employed in each assay. These results demonstrated conclusively that we can now have a constant source of effector cells and target cells, which can be used from assay to assay as an internal standard.     

Sudden incapacitation: USAF experience, 1970-80

During the period 1970-80, there were reported 146 cases of in-flight sudden incapacitation in the USAF. Of these, 62 involved pilots, 14 were navigators, and 70 were student pilots. The etiologies of sudden incapacitation included illness without loss of consciousness, loss of consciousness, spatial disorientation, and improper M-1 maneuver. Each of these categories is analyzed with emphasis upon prevention, for example, not flying with symptomatic preexisting disease, continued emphasis upon spatial disorientation training, and correct performance of the M-1 maneuver. Based upon the data, conclusions and recommendations are suggested to minimize the risk of episodes of in-flight sudden incapacitation.     

Adaptive resynthesis of O6-methylguanine-accepting protein can explain the differences between mammalian cells proficient and deficient in methyl excision repair

Mammalian cells have been classified as proficient (Mer(+)) or deficient (Mer(-)) in methyl excision repair in terms of their cytotoxic reactions to agents that form O(6)-alkylguanine and their abilities to reactivate alkylated adenoviruses. O(6)-Methylguanine (O(6)MeGua) is considered to be a lethal, mutagenic, and carcinogenic lesion. We measured the abilities of cell extracts to transfer the methyl group from an exogenous DNA containing O(6)MeGua to acceptor protein. The constitutive level of acceptor activity was independent of the Mer phenotype and was approximately 100,000 acceptor sites per cell. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) results in a dose-dependent decrease in the acceptor activity in extracts because the rapid reaction between endogenous O(6)MeGua and acceptor protein makes the latter unavailable for further reaction. Treatment of cells with 1 muM MNNG for 15 min or 2 muM for approximately 2 min uses up >95% of the constitutive activity. However, Mer(+) cells, which are resistant to MNNG, rapidly resynthesize new acceptor protein, and the activity returns to the basal level in approximately 90 min. In Mer(-) tumor cells and Chinese hamster cells, which are sensitive to MNNG, resynthesis is not detectable in 90 min. Mer(-) simian virus 40-transformed fibroblasts, known to have an intermediate sensitivity to MNNG, have an intermediate resynthesis rate. Treatment of cells with multiple low doses of MNNG results in the enhanced production of O(6)MeGua-accepting protein in levels 2.5-fold above the constitutive values for Mer(+) tumor cells and to approximately 1.5-fold for Mer(+) fibroblasts or Mer(-) simian virus 40-transformed cells. Such treatments reduce the activities in Mer(-) tumor cells and Chinese hamster cells. We conclude: (i) estimates of O(6)MeGua in cellular DNA shortly after treatment may be seriously in error because of the rapid repair of this lesion, and (ii) the adaptive resynthesis of acceptor protein, not its constitutive level, is the important correlate of cell resistance to methylating agents.     

A 3-degree of freedom binary search pose estimation technique

This paper describes a novel, binary search pose estimation (BSPE) technique for poses constrained to 3 degrees of freedom (DOF). The technique requires three fiduciary marker points and operates by minimising the angular DOF through a binary search driven algorithm. The technique is less computationally intensive than the standard closed form solutions and quickly converges to a solution which can be verified if additional fiduciary points are available. Evaluation for the technique is presented through a comparison to pose from orthography and scaling with iterations (POSIT) using synthetic data and a real-world application, where the technique is used for pose estimation of a mobile robot with respect to a motor vehicle for inspection purposes.     

Growth and feeding efficiency of wild and aquaculture genotypes of rainbow trout (Oncorhynchus mykiss) common to Lake Huron,Canada

Selective breeding of salmonid fishes for the purpose of commercial aquaculture has resulted in domesticated strains possessing a divergent physiological and behavioral phenotype from that of wild conspecifics. Freshwater production of rainbow trout (Oncorhynchus mykiss) has been occurring in regions of Lake Huron, Canada, for decades yet the growth and performance of domestic (aquaculture) versus wild (naturalized) strains are poorly understood. We conducted two trials to examine growth differences between size-matched wild and domestic strains of juvenile rainbow trout: (1) reared separately and fed to satiation; and (2) reared together and fed a reduced ration to induce competition. Additionally, we used bioenergetics models to assess strain-specific growth rates across a range of water temperatures (5, 10, 15 and 18 °C) as well as Lake Huron temperatures during the open-water season. Domestic rainbow trout showed a growth advantage throughout the 102 d trials, and by the end of the study had achieved a mass > two-fold that of the wild strain and had greater fork length, condition, and thermal growth coefficient (TGC) under both treatments. Rapid growth of domestic strain fish was achieved through the combination of enhanced feed consumption (by ~ 40%) and feeding efficiency (up to 60% lower feed conversion ratio) relative to wild fish. Divergence in growth rates between strains was most pronounced (> 3 ×) when modeled with Lake Huron open-water temperatures. We demonstrate that the growth and feed-conversion efficiency differ significantly between these two strains of rainbow trout under laboratory conditions, suggesting that differences could be even greater in nature.     

Effects of phentermine on responding maintained under multiple fixed-ratio schedules of food and cocaine presentation in the rhesus monkey

Drugs that decrease drug-maintained responding at doses that do not decrease other behaviors in animals may be suitable candidates for development as medications to treat drug abuse in humans. The present study examined whether this effect could be obtained with phentermine, a drug that has been reported to decrease cocaine intake in humans. Rhesus monkeys were trained under multiple fixed-ratio 30-response schedules of food and i.v. cocaine delivery. Phentermine was always given as a slow, i.v. infusion. Acute treatment with phentermine (0.3-10 mg/kg) decreased cocaine-maintained responding at doses that did not decrease, or decreased less, food-maintained responding for each of three unit doses of cocaine (10-100 microg/kg/injection). Subacute treatment with phentermine (3 or 5.6 mg/kg, daily) also decreased cocaine-maintained responding more than food-maintained responding. After subacute treatment was terminated, rates of cocaine-maintained responding generally recovered to levels comparable to those seen during untreated control sessions. Phentermine (0.3-3 mg/kg) did not generally increase responding associated with a very low (1 microg/kg/injection) unit dose of cocaine, suggesting that the decrease in cocaine-maintained responding at higher unit doses was not the result of a leftward shift in the cocaine unit dose-effect function. Phentermine (0.1-3 mg/kg) decreased responding maintained by 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909) (30 microg/kg/injection) at doses similar to those that decreased food-maintained responding. These results show that phentermine is effective in decreasing cocaine self-administration and suggest that it may be an effective medication for cocaine abuse.