A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane.

Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370-1394 and 1404-1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.

Diamide-induced cytotoxicity and thermotolerance in CHO cells.

Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide-denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0 degrees C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees C [Freeman et al., J. Cell. Physiol., 164:356-366 (1995); Senisterra et al., Biochemistry 36: 11002-11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.

The Intensity of Home Care Acuity Scale. Part one.

Competition and consumer demand for lower-cost health services are affecting how patients receive home care. To help support field staff, one agency created an quantitative tool--Intensity of Home Care Acuity Scale.

Thermotolerance expression in mitotic CHO cells without increased translation of heat shock proteins.

The objective of this study was to unequivocally demonstrate thermotolerance expression in mammalian cells in the absence of stress-induced synthesis of heat shock proteins (HSPs). Mitotic cells were selected as an experimental system since their genome was in the form of condensed chromosomes and ostensibly incapable of being transcribed; thus, obviating stress-induced HSP gene expression. Asynchronous Chinese hamster ovary (CHO) cells were treated with 0.2 microgram/ml nocodazole to accumulate cells in mitosis for harvest by mitotic shakeoff. Cells were maintained in mitosis with nocodazole during thermotolerance induction, thermotolerance development, and all challenge hyperthermia exposures. Although the heat shock transcription factor was activated by the thermotolerance inducing heat shock, as indicated by gel mobility shift assay, no increase in steady-state HSP mRNA levels was detected, as expected. Preferential synthesis of HSPs from extant mRNA was not detected during thermotolerance development and cellular levels of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins remained constant, as determined by Western Blot analyses. The magnitude and induction threshold of expressed thermotolerance was not diminished when cells were incubated with 10.0 micrograms/ml cycloheximide during thermotolerance development confirming that new protein synthesis was not requisite. Parallel experiments were performed using nonmitotic cells in which protein synthesis was inhibited during thermotolerance development with 10.0 micrograms/ml cycloheximide. As with mitotic cells, high levels of thermotolerance were attained without detectable increases in the cellular content of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins. The results of this study demonstrated that high levels of thermotolerance could be expressed in mitotic cells without stress-induced, preferential synthesis of HSPs, and support the contention that a substantial fraction of thermotolerance expressed in nonmitotic cells also occurs independently of induced HSP synthesis.

Characterization of a signal generated by oxidation of protein thiols that activates the heat shock transcription factor.

The diazenecarbonyl derivative, diamide, was used to produce nonnative protein disulfides in Chinese hamster ovary cells in order to characterize the events that occur during thiol oxidation-induced denaturation that trigger induction of Hsp 70. We limit the term protein denaturation to a process involving a conformational rearrangement by which the ordered native structure of a protein changes to a more disordered structure. Protein thiol oxidation resulted in immediate destabilization of proteins, as assessed by differential scanning calorimetry (DSC). The DSC profile indicated both a decrease in the onset temperature for detection of denaturation and destabilization of a class of proteins with an average transition temperature (Tm) of 60 degrees C. Concomitant with destabilization was an increase in proteins associated with isolated nuclei. Thiol oxidation also induced heat shock transcription factor (HSF) binding activity, however, this was nearly undetectable immediately following diamide treatment: maximum activation occurred 3 hr following exposure. In contrast, heat shock denatured thermolabile proteins which exhibited a Tm of < or = 48 degrees C. Heat shock also resulted in a rapid increase in proteins associated with isolated nuclei and produced immediate and maximum activation of HSF binding. The accumulation of Hsp and Hsc 70 mRNA following thiol oxidation reflected the delay in HSF binding. Acquisition of HSF binding activity occurred immediately if diamide-treated cells were subsequently exposed to a heat shock, indicating that HSF was not inactivated by the diamide treatment. Ostensibly, the cellular system for detecting denatured/abnormal proteins failed to immediately recognize the signal generated by thiol oxidation. These results suggest that at least two processes are involved in the induction of Hsp 70 by nonnative disulfide bond formation: destabilization of protein structure resulting in denaturation and recognition of denatured protein.

Protocol for freezing thermotolerant cells and maintaining thermotolerance following thawing.

Two independent laboratories have demonstrated that suspension-grown, Chinese hamster ovary (CHO) cells can be made thermotolerant, frozen and subsequently thawed such that they still express thermotolerance. Thermotolerance was determined as the ability to protect cells against hyperthermic cell killing (colony formation assay) and the ability to reduce protein aggregation within the nuclei of heated cells. Cells were frozen either following development of full or partial thermotolerance. In the former case frozen cells maintained thermotolerance upon thawing and in the latter case cells subsequently developed full thermotolerance following thawing and incubation at 37.0 degrees C. After thawing, frozen cells displayed a temporal course of thermotolerance development and decay that was similar to that for never-frozen cells. Success was obtained using either asynchronous or synchronous cell populations, and the heat sensitivity of the cells was not altered by the freezing procedure. The experimental results demonstrate the plausibility of utilizing a frozen stock of thermotolerant cells to make thermotolerance experiments more convenient.

Modification of tumour glucose metabolism for therapeutic benefit.

Tumours have a much greater dependence than normal tissues on anaerobic glycolysis for energy generation. We have studied the effects of glycolysis inhibition on tumour cells in vitro. Cellular ATP fell during exposure of cells in air to 2-deoxy glucose or to the lactate dehydrogenase inhibitor oxamate. Glycolysis inhibition alone did not alter clonogenic cell survival over 6 h, but a 6-h exposure to oxamate combined with doxorubicin (Dox) gave greater than additive cell killing. This effect was greatest when oxamate was dosed after Dox, suggesting that oxamate inhibited repair of Dox-induced damage. Oxamate also gave greater than additive cell killing in multicellular spheroids when combined with Dox and there was a greater than additive growth delay in spheroids dosed with Dox plus oxamate. These data demonstrate that inhibition of anaerobic glycolysis might be used to obtain a significant therapeutic gain in combination treatments with cytotoxic drugs or radiotherapy.

Induction of tolerance to hypothermia and hyperthermia by a common mechanism in mammalian cells.

Pretreatment by hypothermic (25 degrees C) cycling (PHC) of attached exponential-phase V79 Chinese hamster cells by Method 4 (24 hr at 25 degrees C + 1.5 hr at 37 degrees C + 24 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) or by Method 3 (48 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) make mammalian V79 cells significantly more resistant to 43 degrees C hyperthermia. There is no significant difference in the 43 degrees C curves whether Method 3 or 4 is used for pre-exposure. If pre-exposure at 15 or 10 degrees C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5 degrees C and, to a lesser extent, to 10 degrees C. The increase in hyper- and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat-shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37 degrees C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10 degrees C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia.

Effect of thermotolerance on heat-induced excess nuclear-associated proteins.

Earlier studies reported that thermotolerance had two effects on the heat-induced increase in nuclear-associated proteins (NAPs); reduction in NAP levels immediately following hyperthermia and facilitation of NAP recovery to control levels. It has also been demonstrated that there are two phases of thermotolerance; one that requires newly synthesized proteins (protein synthesis dependent thermotolerance; PSDT), and another that does not (protein synthesis independent thermotolerance; PSIT). This study was designed to determine if these two phases of thermotolerance affected NAP binding in a similar or different manner. The results demonstrated that protein synthesis during thermotolerance development was not required to reduce NAP levels measured immediately following hyperthermia, but was required to facilitate NAP recovery to control levels following hyperthermia. Reducing NAP levels was the predominant mechanism by which thermotolerance protected cells from this lesion at 43.0 degrees C while facilitated NAP recovery predominated in protecting against exposure to 45.5 degrees C. The facilitated recovery of NAPs required only proteins synthesized following thermotolerance induction and prior to the second heat challenge. Proteins synthesized following the second heat challenge were not requisite. Finally, the processes that facilitate NAP recovery were inhibited at 3 degrees C, suggesting that they are enzymatically mediated.

Cycloheximide protection against actinomycin D cytotoxicity.

Pretreatment plus concomitant treatment with 10 micrograms/ml cycloheximide protected Chinese hamster ovary cells and Swiss 3T3 cells against the cytotoxicity of actinomycin D. The cycloheximide treatment reduced the intracellular concentration of actinomycin D by reducing the level of actinomycin D bound to the acid precipitable fraction of the cell. Levels of unbound actinomycin D were unaffected by cycloheximide, indicating that the plasma membrane permeability to AD was not reduced. Actinomycin D inhibited total transcription but did not reduce cytoplasmic levels of rRNA nor of most tested mRNA; however, cytoplasmic levels of c-myc mRNA were reduced below detectability. Cycloheximide treatment further inhibited total transcription and had no effect on cytoplasmic levels of rRNA nor of most tested mRNA. Cytoplasmic levels of c-myc were elevated by cycloheximide and remained so even in the presence of actinomycin D. These data suggested that a reduction in cytoplasmic levels of short lived, essential mRNA, such as c-myc mRNA, was one lethal lesion of actinomycin D. Furthermore, cycloheximide's protection may result, in part, from its ability to stabilize and/or elevate cytoplasmic levels of these mRNA, thus counteracting their depletion by actinomycin D. Protection may also result from the cycloheximide-induced reduction of actinomycin D bound to the acid precipitable fraction of the cells.

Reduction of levels of nuclear-associated protein in heated cells by cycloheximide, D2O, and thermotolerance.

Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.

Evaluation of the Hemalog D differential leucocyte counter.

The main objectives of the evaluation were to (1) establish 'normal values' for the Hemalog D, (2) compare the Hemalog D with existing manual procedures, and (3) assess the machine's reliability. It proved to be a practical machine in a routine haematology laboratory. Comparison with established techniques showed systematic differences in the total white blood cell count and percentages of lymphocytes. An existing laboratory computer system was used to collect and file the data.

Deoxyuridine suppression test: a comparison of two methods and effect of thymidine on the incorporation of 3H-deoxyuridine into DNA in human bone marrow cells.

Deoxyuridine suppression tests have been performed by two different methods of six normoblastic and eight megaloblastic marrows. A good correlation was obtained between the results by the modified and the original methods. The simplified method was found to be applicable for a clinical purpose to diagnose megaloblastosis in the marrow. Uptake of 3H-deoxyuridine into DNA and effect of various concentrations of thymidine was studied on five normoblastic and six megaloblastic marrows. In megaloblastic marrows, a greater amount of thymidine was required to obtain the same rate of suppression of 3H-deoxyuridine incorporation into DNA than in normoblastic marrows. Impairment of thymidine incorporation into DNA in megaloblastic marrows was not revealed. Therefore, lower rate of suppression of 3H-deoxyuridine by thymidine in megaloblastic marrows may be due to impairment of the incorporation of deoxyuridine before the addition of thymidine.