Temperature-sensitive Process Research Articles

Adenosine 3',5'-monophosphate modulates thyrotropin receptor clustering and thyrotropin activity in culture.

A biologically active rhodamine conjugate of thyrotropin binds at 4 degrees C to diffusely distributed membrane thyrotropin receptors which patch and become endocytosed into thyroid cells in a temperature-sensitive process. When the cells are first incubated with 8-bromo-cyclic adenosine monophosphate at 37 degrees C, the conjugate also binds to clustered receptors at 4 degrees C. Furthermore, 8-bromo-cyclic adenosine monophosphate reduces the amount of adenosine 3',5'-monophosphate (cyclic AMP) induced by thyrotropin. Hence, increased intracellular cyclic AMP induces receptor patching and reduces the concentration of cyclic AMP normally induced by thyrotropin. This suggests that cyclic AMP acts both as the second messenger of thyrotropin and also as the regulator of the level of thyrotropin receptors.

Metabolic characteristics of renal insulin uptake.

The kidney removes insulin from the circulation by glomerular and peritubular clearance, thus exposing both the luminal and the contraluminal tubular surfaces to the hormone. In the proximal tubule, filtered insulin is internalized by means of pinocytosis and eventually localizes in the lysosomes. The fate of insulin removed from the peritubular circulation is less well understood. We previously reported that tubular uptake of insulin is a temperature-sensitive process consisting of two different systems. The system associated with the luminal aspect of the cell appears to be dependent on oxidative metabolism, whereas the system associated with the contraluminal aspect of the cell appears to be independent thereof. To further characterize renal tubular insulin uptake, a study was performed with (OCI) was 1336 ± 51 μl/min (SEM), glomerular filtration rate (GFR) was 1019 ± 54 fillmin, peritubular clearance of insulin was 317 ± 56 μl/min, fractional excretion of sodium (FEna) was 5.1 ± 0.7%, and fractional excretion of insulin (FEi) was 1.3 ± 0.6%. Addition of inhibitors of glycolysis (iodoacetate), Na-K-ATPase (ouabain), and lysosomal degradation (chloroquine) was associated with a significant increase of Fei (4.7 ± 0.4%, 6.4 ± 1.4%, and 13.9 ± 2.4%, respectively) and of FEna (28%, 43%, and 12%, respectively). By contrast acetazolamide with furosemide increased FEna to 39%, without altering FEi. Analysis of all data revealed no correlation between FEna and FEi, indicating that tubular insulin absorption is at least partly independent of sodium reabsorption. None of the inhibitors altered OCI, GFR, or peritubular clearance significantly. The above observations, together with our previously reported data, indicate that luminal insulin uptake is a temperature and chloroquine sensitive process that is dependent on metabolic energy. The source of this energy appears to be ATP, which is derived from oxidative metabolism and glycolysis. These characteristics, consistent with a process involving pinocytosis and lysosomal degradation, were not features of the contraluminal insulin uptake process.

Effects of high temperature on the germination of maize (Zea mays L.)

Poor emergence of maize seedlings, due to high soil temperatures, is a major limitation of crop potential in the lowland tropics. Ability to germinate at high temperature (>c. 37° C) is related to the temperature sensitivity of the embryo, and there is considerable genotypic variation for this character.Respiration and mitochondrial phosphorylation proceed normally in seeds imbibing at 41° C, and ATP levels are adequate for germination. However, the specific activities of several important enzymes are lower, and the rate of protein synthesis is severely reduced compared with seeds imbibing at 28° C. The depression of the rate of protein synthesis in the embryos of several tropical hybrids imbibing at high temperature correlated with their known temperature sensitivity. It is concluded that protein synthesis is an especially temperature sensitive process in germinating maize embryos, and that this is the principal reason for the sensitivity of germinating maize seeds to high temperature.

Inhibition of synaptosomal choline uptake by tetanus and botulinum A toxin. Partial dissociation of fixation and effect of tetanus toxin.

Tetanus toxin and, to a lesser degree, botulinum A toxin inhibit partially and noncompetitively the uptake of [3H]choline into a crude synaptosomal fraction from rat brain cortex. Botulinum toxin acts by its neurotoxin content. The effect is not due to nonspecific synaptosomal damage by the toxins as shown by the lactate dehydrogenase occlusion test, by the absence of swelling and by the preservation of choline stores. The ratio between [3H]acetylcholine and [3H]choline was decreased by both toxins. Inhibition by either toxin depends strongly on the temperature and duration of incubation, and is preceded by an initial latency period. The effect of tetanus toxin, once manifest, is largely resistant against antitoxin. It is not significantly diminished by pretreatment of the synaptosomes with V. cholerae neuraminidase. Fixation of 125I-tetanus toxin proceeds fast, is largely independent of temperature and is diminished by pretreatment of the synaptosomes with neuraminidase. Thus only some of the fixation sites, and not the long-chain gangliosides, are required for the effects of tetanus toxin. A slow, temperature-sensitive process links the fixation with the action. In contrast to rat synaptosomes the chicken preparation is more sensitive to botulinum A than to tetanus toxin, which reflects the differences in sensitivity between live birds and rodents. Our data underline the similarities between the effects of tetanus and those of botulinum A toxin. Their dependence on time and temperature, the time dependence of efficacy of antitoxin, and the concordance in species specificity indicate that the in vitro system mirros some crucial features of poisoning of isolated organs and live animals.

Internalization and intracellular fate of Wistaria floribunda agglutinin in BALB/c 3T3 fibroblasts

The accumulation of large lysosomes in BALB/c 3T3 fibroblasts following exposure of the cells to Wistaria floribunda agglutinin (WFA) was accompanied by adsorptive endocytosis of the lectin. The internalization of WFA was examined by a double label immunofluorescence technique that could distinguish internal WFA from cell surface WFA in the same cell. The lectin bound to the plasmalemma of the cell via its saccharide binding site resulting in the aggregation into a surface cap of some lectin-receptor complexes. Lectin was internalized by a temperature-sensitive process. The lectin accumulated within the cell over a 24 h period as shown by an increase with time of the [ 125I]WFA that could not be washed from the cells by the competitive saccharide inhibitor, lactose. The vesicles containing WFA combined with lysosomes as evidenced by (1) the localization of ferritin-WFA conjugates in vesicles having the size and morphology of lysosomes; and (2) the degradation of radiolabeled lectin to an ethanol-soluble form. Lectin treatment had no significant effect on the overall degradation of intracellular proteins in growing or serum-starved cells. It is suggested that WFA either enhances the rate of fusion of lysosomes with other lysosomes or lectin-containing endocytic vesicles or inhibits the process by which lysosomes turn over.

Uptake and Transport of Fusicoccin in Higher Plant Tissues

The uptake and release of 3H-labelled fusicoccin (FC) fed to pea internode segments and the transport of [3H]FC in whole plants and in excised parts of plants of pea, maize, and pumpkin have been investigated. FC uptake showed three phases apparently corresponding to the pene tration of FC: (1) into the free space; (2) into the cytoplasm; (3) into the vacuole. The first phase is a little thermosensitive and apparently includes adsorption binding to some free space component (cell surface?). The second and third phases are highly temperature sensitive and in part energy-dependent. However, no accumulation of FC against a concentration gradient is observed at high external FC concentration. Accumulation at low concentration seems due to the formation of a poorly exchangeable complex with some cell structure. The efflux of the FC penetrated within the cells is limited by some highly temperature sensitive process. A small fraction of the accumulated FC tends to be retained in the tissue, either because sequestered in some compartment or because bound to some cellular component. FC transport in whole plants as well as in isolated plant parts seems to depend mainly on simple diffusion and on transport by mass flow in the xylem. Long distance transport of the toxin in pumpkin plants mainly occurs in the xylem, while the slow movement in non-vascular tissues seems to depend on diffusion combined with mass flow in the free space, and does not seem heavily influenced by metabolic factors.

Interaction of tritium-labeled H2DIDS (4,4′-diisothiocyano-1,2,diphenyl ethane-2,2′disulfonic acid) with the ehrlich mouse ascites tumor cell

The experiments reported in this paper were undertaken to explore the interaction of tritiated H2DIDS (4,4'-diisothiocyano-1,2,diphenyl ethane-2,2'-disulfonic acid) with Ehrlich ascites tumor cells. Addition of (3H)H2DIDS to tumor cell suspension at 21 degrees C, pH 7.3, resulted in: (i) rapid reversible binding which increased with time and (ii) inhibition of sulfate transport. Tightly bound H2DIDS i.e., reagent not removed by cell washing, also increased with time. Binding of 0.02 nmol H2DIDS/mg dry mass or less did not affect sulfate transport, but, at greater than 0.02 nmol and up to 0.15 nmol the relationship between tight binding and inhibition of transport is linear. The fact that H2DIDS could bind to the cell and yet not affect anion transport suggests that binding sites exist unrelated to those concerned with the regulation of anion permeability. Support for this is the observation that H2DIDS is spontaneously released from cells even after extensive washings by a temperature-sensitive process. The most important source of released H2DIDS is the cell surface coat which labels rapidly (within 1 min) and is then spontaneously released into the medium. A second source is derived from H2DIDS that slowly entered the cells. Consequently, at least four modes of interaction exist between H2DIDS and ascites tumor cells. These include both reversible and irreversible binding to membrane components which regulate anion permeability, irreversible binding to cell surface proteins or glycocalyx, and finally incorporation of H2DIDS into the intracellular phase.

Effects of glucocorticoids on fibroblasts.

Mouse fibroblasts growing in vitro respond to glucocorticoids in a dose-dependent fashion by reduced rates of growth. The inhibition of growth observed in vitro is related to the topical anti-inflammatory action of glucocorticoids and to their capacity to inhibit wound repair. The cells growing in vitro possess a glucocorticoid receptor system that has been studied in some detail using [3H]triamcinolone acetonide as a radiolabeled ligand. The initial binding reaction occurs in the cytosol. The complex is then rapidly taken up in the nucleus of the cell by a temperature-sensitive process. In the nucleus, the complex exists in two forms, one of which is readily extracted by 0.3 M KCl solutions. A small amount of steroid-receptor complex is tightly bound to chromatin. Under normal incubation conditions, there is a constant cycling of steroid-receptor complex, and unbound receptor is generated back into the cytosol from the nucleus with a half-life of about 30 min. Regeneration of unbound receptor does not depend on protein synthesis and is a temperature-sensitive and energy-requiring process. Incubating the steroid-treated cells in the absence of glucose and in the presence of inhibitors such as cyanide or dinitrophenol leads to a loss of cytoplasmic steroid-receptor complexes, and an accumulation of the complex in the nuclear residual form, tightly bound to chromatin. With respect to nuclear effects of steroid treatment, we have found that incubating fibroblasts in vitro with glucocorticoids produces a prompt decrease in the amount of a satellite H1 histone found in these cells.

Dormancy studies in seed of Avena fatua. 9. Demonstration of genetic variability affecting the response to temperature during seed development

Experiments are reported which demonstrate characteristic differences among pure lines of wild oats in the degree to which seed dormancy is influenced by temperature during seed development. In some lines, high temperature during seed maturation greatly reduced the duration of primary dormancy; in others, this effect was relatively slight. Evidence is discussed that two temperature-sensitive physiological processes influence the duration of dormancy in this species.

Factors influencing the handling of insulin by the isolated rat kidney.

The renal handling of immunoreactive insulin was studied in the isolated perfused normothermic rat kidney to determine (a) the relative contributions of glomerular clearance and peritubular clearance to the renal clearance of insulin under different conditions, (b) what metabolic factors influence the ability of tubular cells to remove insulin from the glomerular filtrate and the peritubular circulation, and (c) whether the same factors influence the luminal and contraluminal uptake of insulin.In control kidneys the organ clearance of insulin (OCi) was 974+/-63 mul/min (SEM), of which a maximum of 46% could theoretically be accounted for by filtration. OCi was not altered by fasting, lack of exogenous fuel (glucose), or the addition of cyanide. The glomerular filtration rate did not correlate with the OCi, but there was a significant (P < 0.001) negative correlation (r = -0.828) between the peritubular clearance and glomerular filtration rate. Both N-ethylmaleimide and cold (10 degrees C) reduced the rate of insulin removal. Fractional excretion of filtered insulin (9.7+/-1.7% in controls) was not significantly altered by fasting or perfusing without glucose. In contrast, KCN increased fractional excretion of insulin to 41.9+/-3.7% whereas cold increased fractional excretion to 69.0+/-3.3%. This study indicates that renal tubular cells remove insulin from the tubular lumen and the peritubular compartment. Furthermore, the data suggest that insulin removal by tubular cells is a temperature-sensitive process consisting of two different systems. The system associated with the luminal aspect of the cell appears to be dependent on oxidative metabolism, whereas the system associated with the contraluminal aspects of the cell appears to be independent thereof. Under several circumstances when the glomerular clearance of insulin falls thereby reducing the amount of insulin absorbed by the luminal aspect of the cell, contraluminal uptake increases, and a constant rate of insulin removal is maintained by the kidney.

Temperature‐sensitive aspects of evoked and spontaneous transmitter release at the frog neuromuscular junction.

1. The temperature dependence of presynaptic processes involved in neuromuscular transmission was studied by rapidly increasing the temperature of cooled frog neuromuscular junctions by 4--10 degrees C using pulses from a neodymium laser. The temperature elevation was complete within 0.5 msec, and decayed back to control levels with a time constant of about 7--8 sec. 2. Temperature jumps completed before nerve stimulation increased the quantal content and decreased the latency of the end-plate potential (e.p.p.). The Q10 for e.p.p. quantal content in low [Ca2+] Ringer averaged about 3.9 over the range 1--18 degrees C. 3. Temperature jumps occurring during the synaptic delay (the interval between the presynaptic action potential and the onset of the e.p.p.) also increased the quantal content and decreased the latency of the e.p.p. These effects diminished as the onset of the temperature jump was moved closer to the expected onset of the e.p.p. Temperature jumps applied after the onset of the e.p.p. immediately accelerated the time course of the e.p.p. but did not significantly alter quantal content. These results demonstrate that the magnitude and timing of evoked release are influenced by temperature-sensitive processes that operate both during and shortly after the presynaptic nerve action potential, but are largely complete before the onset of release. 4. Temperature jumps were applied at various times during the interval between two nerve stimuli. The amplitude of the second e.p.p. decreased as the temperature jump was moved earlier in the interstimulus interval, suggesting that the rise in temperature following the first nerve stimulus accelerates the decay of facilitation. When the temperature jump was moved from 10 msec after to 10 msec before the onset of the first e.p.p., the amplitude of the second e.p.p. either decreased or showed no change. The fact that the second e.p.p. did not increase suggests that the temperature-sensitive processes that increase the quantal content of the conditioning e.p.p. do not greatly increase the facilitation following that e.p.p. 5. Temperature jumps immediately accelerated the time course of spontaneous miniature end-plate potentials (m.e.p.p.s) and increased their frequency. Experiments using slow temperature changes revealed that the Q10 for m.e.p.p. frequency in normal Ringer is about 10 over the range 10--20 degrees C. M.e.p.p. frequency was much less sensitive to temperature changes below about 10 degrees C. When the nerve terminal was depolarized by 20 mM-K+ in the presence of Ca2+, the Q10 for the rate of spontaneous release over the range 10--20 degrees C decreased to about 4, similar to the Q10 for e.p.p. quantal content. In the absence of extracellular Ca2+ the Q10 for m.e.p.p. frequency in 20 mM-K+ remained near 10. 6. The marked difference in Q10S for spontaneous transmitter release under different experimental conditions suggests that not all transmitter release uses identical mechanisms...

A conditional lethal mutant of Escherichia coli which affects the processing of ribosomal RNA.

A temperature-sensitive mutant strain was isolated from an RNase III-(rnc) strain of Escherichia coli. At the permissive temperature it behaves like the parental strain, but at the nonpermissive temperature it fails to produce normal levels of 23 S and 5 S rRNA, while instead the 25 S rRNA species becomes very prominent. (The 25 S molecule appears in rnc cells and contains 23 S rRNA sequences). When an rnc+ mutation was introduced to such a strain, or when the rnc mutation was replaced by an rnc+ allele, the strain remained temperature-sensitive. At the permissive temperature such strains synthesized rRNA like any other E. coli strain, but at the nonpermissive temperature they remained unable to synthesize normal levels of 5 S rRNA, and instead a larger molecule was accumulated. The simplest interpretation of theses findings is that the mutant strain contains a temperature-sensitive processing endoribonuclease, RNase E, which normally introduces a cut in the growing rRNA chain somewhere between the 23 S and the 5 S rRNA cistrons. These findings help also to explain the nature and origin of the various rRNA species observed in RNase III- cells and add to our understanding of processing of ribosomal RNA in normal cells of Escherichia coli.

The thermal history of a spot heated by a laser

A numerical solution of the thermal history of a spot heated by a laser is presented. The laser is assumed to be operated with a coaxial nonreacting gas jet as is normal practice when surface hardening, or surface alloying by vapour deposition. The solution is obtained by a Crank-Nicholson method and assumes convective and radiant surface losses. It is presented in the form of graphs from which surface temperature profiles can be calculated by hand. The solutions show that the cooling due to the gas jet and surface radiant losses can not be ignored when considering temperature sensitive processes.

Abnormalities in somite segmentation following heat shock to Xenopus embryos

The typical abnormality induced by a 15 min shock at 37degreesC is a single discrete length along the somite file within which segmental boundaries are absent or irregular. The two sides of the same embryo present a similar but not necessarily identical appearance. Usually all the embryos in a treated batch show abnormalities of similar severity. Survival of treated embryos, the details of the visible malformations, and temporal aspects of the phenomenon have been studied. The results indicate a temperature sensitive period that traverses the neurula, from head to tail at about the same rate as the somites form, but some hours beforehand. The temperature sensitive process is not associated with cell determination and differentiation, and there are reasons for thinking that the specification of the normal somite number occurs independently. The results are discussed in relation to Cooke & Zeeman's model of a wave front interacting with an oscillator.

Responses of hair cells in the statocyst of Hermissenda.

Responses to mechanical stimulation were recorded from hair cells in the statocyst of Hermissenda crassicornis. The response to a brief stimulus is a depolarizing wave which reaches peak in about 25 msec and decays slowly. 2. Hyperpolarization by extrinsic currents increases the amplitude of the response; depolarization decreases it and eventually reverses its polarity. It is inferred from these results that the primary outcome of the transduction process is an increase of membrane conductance and that the voltage change (generator potential) follows as a secondary event. 3. The features of the conductance change were reconstructed from the time course of the generator potential and the passive properties of the membrane. It was found that the increase of membrane conductance develops slowly and is roughly proportional to the energy delivered by the stimulus. 4. The time course of the conductance change required to reproduce the generator potential is similar to the output of a model involving a sequence of transformations. 5. The generator potential is sensitive to temperature, becoming faster as temperature is raised. This effect is reproduced by the model if the transition rates are assumed to be temperature-dependent, with a Q10 of about 2. 6. It is concluded that a chain of temperature-sensitive processes is interposed between the stimulus and the increase of membrane conductance.

Specific prostaglandine E 1 and A 1 binding sites in rat a dipocyte plasma membranes

Rat adipocyte plasma membranes sacs have been shown to be a sensitive and specific system for studying prostaglandin binding. The binding of prostaglandin E 1 and prostaglandin A 1 increases linearly with increasing protein concentration, and is a temperature-sensitive process. Prostaglandin E 1 binding is not ion dependent, but is enhanced by GTP. Prostaglandin A 1 binding is stimulated by ions, but is not affected by GTP. Discrete binding sites for prostaglandin E 1 and A 1 were found. Scatchard plot analysis showed that the binding of both prostaglandins was biphasic, indicating two types of binding sites. Prostaglandin E 1 had association constants of 4.9 · 10 9 1/mole and 4 · 10 8 1/mole, while the prostaglandin A 1 association constants and binding capacities varied according to the ionic composition of the buffer. In Tris-HCl buffer, the prostaglandin A 1 association constants were 8.3 · 10 8 1/mole and 5.7 · 10 7 1/mole, while in the Krebs—Ringer Tris buffer, the results were 1.2 · 10 9 1/mole and 8.6 · 10 6 1/mole. Some cross-reactivity between prostaglandin E 1 and A 1 was found for their respective binding sites. Using Scatchard plot analysis, it was found that a 10-fold excess of prostaglandin E 1 inhibited prostaglandin A 1 binding by 1–20% depending upon the concentration of prostaglandin A 1 used. Prostaglandin E 1 competes primarily for the A prostaglandin high-affinity binding site. Similar Scatchard analysis using a 20-fold excess of prostaglandin A 1 inhibited prostaglandin E 1 binding by 10–40%. Prostaglandin A 1 was found to compete primarily for the E prostaglandin low-affinity receptor. All of the bound [ 3H]prostaglandin E 1, but only 64% of the bound [ 3H]-prostaglandin A 1 can be recovered unmetabolized from the fat cell membrane. There is no non-specific binding of prostaglandin E 1, but 10–15% of prostaglandin A 1 binding to adipocyte membranes is non-specific. Using a parallel line assay to measure relative affinities for the E binding site, prostaglandin E 1 > prostaglandin A 2 > prostaglandin F 2α. Prostaglandin E 2 and 16,16-dimethyl prostaglandin E 2 were equipotent with prostaglandin E 1, while other prostaglandins had lower relative affinities. 7-Oxa-13-prostynoic acid does not appear to antagonize prostaglandin activity in adipocytes at the level of the receptor.

THE TIMING OF TEMPERATURE SENSITIVITY OF LEAKY FERTILITY FACTOR MUTATIONS IN MALE DROSOPHILA MELANOGASTER

Some of the mutations of the male fertility factors on the Y chromosome give rise to a small proportion of fertile males, each of which produces only a small number of progeny. Many of these mutations are temperature-sensitive, producing a larger proportion of fertile males at 25 degrees C than at 19 degrees C. One of these mutants was carefully analyzed and it was found that only sperm which mature in the first five days of adult life can effect fertilization, regardless of the temperature regimen. A unique temperature-sensitive period was localized to the mid-pupal stage by single twenty-four-hour 25 degrees C treatments applied at various times in the life cycle to flies otherwise grown at 19 degrees C. The temperature sensitivity associated with this pupal sensitive period is adequate to account for all of the difference in fertility between flies grown continuously at the two temperatures. The timing of the temperature-sensitive process in combination with a cytological study of spermiogenesis indicates that temperature sensitivity occurs after the first meiotic metaphase during a spermatid stage. Thus it affects some post-transcriptional event in the expression of the phenotype of the fertility factor.

Uptake of chondroitin sulfate by mammalian cells in culture: II. Kinetics of uptake and autoradiography

The incorporation of 35 S-labelled chondroitin sulfate ( 35 S-Ch) into cultured Chinese hamster cells was studied quantitatively. 35 S-Ch uptake is characterized as a function of the incubation time, the concentration of 35 S-Ch in the medium, and is dependent on pH and temperature of the incubation medium. The retention of incorporated 35 S-Ch is also a temperature-sensitive process. Autoradiographic studies showed the cytoplasmic localization of the ingested 35 S-Ch.

Characterization of a temperature-sensitive mutant of polyoma virus

A mutant of polyoma virus (TS-a) which is temperature sensitive in both its plaque-forming and transforming abilities at the nonpermissive temperature of 38.5 °, but not at the permissive temperature of 31.5 ° has been further characterized. TS-a was found to be inhibited in its ability to produce infectious virus in mouse cells at 38.5 °; this inhibition could be overcome by infection with high multiplicites. The ratio of the yield of TS-a to the yield of wild-type polyoma increases from 10 −3 to almost unity when the input multiplicity varies from 0.1 to 100 PFU/cell. The temperature sensitivity of TS-a is due not to an increased heat lability of the structural components (capsid protein and DNA) of the completed virus particles, but to an increased heat sensitivity of some intracellular process in viral development. This temperature-sensitive process has been found to take place after uncoating of the infecting virus particle, occurring at the time of, and being required for, the production of virus-sized DNA; the “induction” of host cell DNA synthesis is not markedly inhibited in TS-a infected resting mouse kidney cells at 38.5 °. This finding implies that the virus must contribute some other function (s) besides causing the “induction” of host cell DNA synthesis for neoplastic transformation to occur (i.e., stimulation of host cell DNA synthesis by itself is not enough to cause transformation). Some of the possible temperature-sensitive processes that could be concerned with both the production of viral DNA and transformation are discussed.

Accumulation of two alkylating agents, nitrogen mustard and busulfan, by murine leukemia cells in vitro

The accumulation of two alkylating agents by murine leukemia cells in vitro was studied. Nitrogen mustard (HN 2) and busulfan rapidly penetrated cells; cell-medium drug distribution ratios of one were established within 3 min at 0° over a wide range of external drug levels. Accumulation of busulfan was not much affected by raising the incubation temperature. NH 2 binding was, however, a remarkably temperature-sensitive process. At10 −5 M levels, busulfan and HN 2 partly inhibited cellular incorporation of precursors into nucleic acids in vitro. When cell types differing widely in sensitivity to these alkylating agents were tested, no differences in capacity for drug accumulation or in subsequent inhibition of nucleic acid synthesis could be found.