Resistance to 1-beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene.

We have recently identified a complementary DNA clone which encodes the complete amino acid sequence for 2'-deoxycytidine kinase (dCK), the enzyme required for the initial phosphorylation of several deoxyribonucleosides and their analogues that are widely used as chemotherapeutic and antiviral agents. In order to identify the molecular basis for dCK deficiency in two clonal T-lymphoblast cell lines generated by virtue of their resistance to 1-beta-D-arabinofuranosylcytosine (ara-C-8D) or to 2',3'-dideoxycytidine (ddC50), we have cloned and sequenced their dCK complementary DNAs. The ara-C-8D cell line contained two identifiable mutations: (a) a 115-base pair deletion within the coding region, corresponding to the fifth exon of the gene and presumably resulting from a splice site mutation; and (b) a G to A point mutation that substitutes glutamic acid for glycine within the ATP-binding domain of the protein. Expression of each protein in Escherichia coli demonstrated a complete loss of catalytic activity and, in the case of the deletion, a proteolytic degradation product of the altered protein. The substitution of a negatively charged amino acid within the ATP-binding domain resulted in loss of enzyme activity with all nucleoside triphosphates tested. The ddC50 cell line contained a single identifiable structural gene mutation in all clones sequenced resulting in the substitution of arginine for glutamine at amino acid 156 of the protein. This mutation markedly diminished the catalytic activity of the expressed protein with the three substrates, deoxycytosine, deoxyadenosine, and deoxyguanosine. On the basis of the presence of a single point mutation and a marked reduction in dCK mRNA in this cell line, we postulate that the second allele either is not expressed or is expressed at extremely low levels. We conclude that cellular resistance to the toxicity of 1-beta-D-arabinofuranosylcytosine and dideoxycytidine in these cell lines is mediated by specific mutations within the dCK gene. Further elucidation of structural genes alterations in dCK-deficient cells will facilitate a more detailed understanding of the functional domains of this complex enzyme.

Posttreatment exposure to camptothecin enhances the lethal effects of x-rays on radioresistant human malignant melanoma cells.

Little is known about the molecular mechanisms responsible for the survival recovery process(es) (known as potentially lethal damage repair), which occurs in mammalian cells following ionizing radiation. Previously, we presented data indicating a role for the DNA unwinding enzyme, topoisomerase I, in DNA repair. We now demonstrate that camptothecin, a specific inhibitor of topoisomerase I, causes dramatic radiosensitization of an extremely resistant human melanoma (U1-Mel) cell line. Camptothecin radiosensitized U1-Mel cells when it was administered either during or immediately following x-irradiation. U1-Mel cells were optimally radiosensitized with 4 microM camptothecin for a period of 4-6 hrs after x-irradiation. Enhanced cell killing by camptothecin was proportional to the initial extent of damage created by x-irradiation; the higher the dose of ionizing radiation, the greater the radiosensitization. The apparent synergy observed with camptothecin and x-rays was irreversible; camptothecin-treated U1-Mel cells were not able to carry out PLDR in a 48 hr period after the drug was removed. We hypothesize that the administration of camptothecin causes lesion modification through a topoisomerase I-mediated mechanism. These data support a role for topoisomerase I in DNA repair and indicate that camptothecin, or more effective derivatives, may have clinical use.

The hsp56 immunophilin component of steroid receptor heterocomplexes: could this be the elusive nuclear localization signal-binding protein?

In many cells, the glucocorticoid receptor undergoes rapid steroid-mediated translocation from the cytoplasm to the nucleus, and this receptor is an excellent model for studying the mechanism of targeted protein movement through the cytoplasm. For such unidirectional movement to occur, the receptor must attach to a retrograde movement system in a manner that involves the nuclear localization signal. It is improbable that such attachment occurs via a direct protein-protein interaction between the receptor and the movement system; rather, one or more linker proteins are likely to be involved. As with other steroid receptors, the glucocorticoid receptor is associated with several other proteins in a heterocomplex. Two of these receptor-associated proteins are the heat shock proteins hsp90 and hsp56, and a third heat shock protein, hsp70, is required for assembly of the receptor heterocomplex. The hormone binding domain of the steroid receptors determines the interaction with both hsp90 and hsp70. Hsp56 is known to bind to hsp90, but its potential site, or sites, of interaction with the receptor are undefined. Hsp56 has recently been cloned and demonstrated to be an immunophilin of the FK506/rapamycin binding class. The immunophilins have peptidyl-prolyl isomerase activity but their cellular functions are unknown. Herein, we review the literature on the hsp56 immunophilin component of the receptor heterocomplex and present a rationale for hsp56 being the protein that determines the direction of receptor movement via a direct protein-protein interaction with the nuclear localization signal.

Corneal foreign bodies--first aid, treatment, and outcomes. Skills review for an occupational health setting.

Eye injuries from foreign body incidents remain prevalent in the workplace setting. Often the professional nurse provides the first line of treatment. The informal class presented at the authors' facility offered a comprehensive, organized presentation of a common injury encountered in the practice of occupational health nursing. Strenghts of the presentation included handouts demonstrating eye eversion technique and a flip chart summarizing the content to be placed in each medical station as quick reference. One challenge involved presenting the information to all nurses. The site encompasses four locations and some nurses function as the only staff in the plant for a given shift. With the support of administration and some creative scheduling, 10 of 17 nurses attended one of three classes offered in one morning, and the remaining 7 were able to view the class on videotape. Videotaping the presentation also provided material for future orientation, as well as an opportunity for review. Overall analysis found this a worthwhile offering relevant to practice. A brief formal written evaluation indicated the objectives for the class were achieved and elicited subjects for future topics. Informal chart reviews to check for documentation of visual acuity testing and eversion of the upper lid for foreign body injuries is another outcome measure currently in progress. In addition, a performance improvement project could be accomplished easily by retrospective chart review of assessment and treatment documentation, and tracking of revisits and referrals. Knowledge of current standards in the assessment, first aid, and treatment of eye injuries is every occupational health nurse's responsibility. However, prevention of foreign body injuries is far superior to any treatment modality available. As highly visible leaders within the occupational setting, nurses can be advocates and role models for safe work practices. Occupational health nurses may promote safe eye practices by actively seeking collaboration with safety departments to continuously monitor and improve eye injury and outcome statistics and use of protective eye-wear. By consistently wearing proper safety eyewear, such as approved goggles or prescription safety glasses with side sheilds, during each and every venture into the work area, occupational health nurses provide a strong role model and have the opportunity to educate employees and encourage safe work practices. It is important to encourage shared responsibility and awareness between workers and management for prevention of foreign body incidents and prompt, accurate treatment when necessary to promote optimal outcome.

Optical contamination measurements on early Shuttle missions.

Two camera/photometers were included as part of the Induced Environment Contamination Monitor on STS-2, -3, -4, and -9 orbital missions to record photographically Shuttle-induced particulate and background contamination. The cameras collected stereoscopic data continuously during the on-orbit phases of these missions making exposures every 150 s. The results recorded throughout a 32 degrees field-of-view indicate high particle concentrations during early mission operations. These decay to a quiescent rate of 500 particles of >10-microm radius observed per orbit. Preliminary size and velocity distributions of measured particles are presented as are measurements of background brightness due to unresolved particles in the visible spectral region.