Monensin blocks the transport of diphtheria toxin to the cell cytoplasm.

Lysosomotropic amines are believed to inhibit the transport of diphtheria toxin to the cell cytoplasm by raising the pH within intracellular vesicles. If so, then other drugs that dissipate intracellular proton gradients should have a similar effect on toxin transport. We found that monensin, a proton ionophore unrelated to lysosomotropic amines, is a potent inhibitor of the cytotoxic effect of diphtheria toxin. Monensin appears to block the escape of endocytosed toxin from a vesicle to the cytoplasm. Monensin fails to protect cells from the effects of diphtheria toxin that is bound to the cell surface and exposed to acidic medium, suggesting that the step normally blocked by the drug is circumvented under these conditions. The inhibition of toxin transport caused by monensin could not be relieved when monensin was replaced by ammonium chloride, nor when ammonium chloride was again replaced by monensin. This suggests that both drugs block the same step of toxin transport. The effect of monensin on the transport of diphtheria toxin to the cytoplasm is consistent with the proposal (Draper and Simon. 1980. J. Cell Biol. 87:849-854; Sandvig and Olsnes. 1980. J. Cell Biol. 87:828-832) that the toxin is endocytosed and then, in response to an acidic environment, penetrates through the membrane of an intracellular vesicle to reach the cytoplasm.

A Chinese hamster ovary cell mutant with a heat-sensitive, conditional-lethal defect in vacuolar function.

We describe a mutant derived from Chinese hamster ovary cells that is offt-sensitive for viability and for resistance to certain protein toxins. This mutant, termed G.7.1, grows normally at 34 degrees C but does not grow in Dulbecco's modified Eagle's medium at 39.5 degrees C. However, when this medium is supplemented with FeSO4, the mutant cells will grow at the elevated temperature. At 39.5 degrees C, G.7.1 cells acquire resistance to diphtheria toxin, modeccin, and Pseudomonas aeruginosa exotoxin A, all of which are protein toxins that require endocytosis and exposure to a low pH within vesicles before they can invade the cytosol and kill cells. The properties of mutant G.7.1 could result from a heat-sensitive lesion that impairs vacuolar acidification. We assayed the ATP-stimulated generation of pH gradients across the membrane of vesicles in cell-free preparations from mutant and parental cells by the partitioning of acridine orange into acidic compartments and found that the acidification response of the mutant cells was heat-labile. Altogether the evidence suggests that G.7.1 cells contain a heat-sensitive lesion that impairs vacuolar acidification and that they fail to grow in normal medium at 39.5 degrees C because they cannot extract Fe+3 from transferrin, a process that normally requires exposing transferrin to a low pH within endosomal vesicles.

Chromosomal analysis of human prostatic adenocarcinoma cell lines.

Although detailed cytogenetic analysis has been carried out in many types of cancer, there is little information on the chromosomal makeup of prostatic cancer cells. Karyological analyses of cell lines derived from both metastatic and primary prostatic carcinoma have been carried out by Q-, C-, and sequential banding techniques. The metastatic line, PC-3, isolated from a bone marrow specimen, is an established epithelial line which is tumorigenic in nude, athymic mice and forms colonies in semisolid agar suspension. A subline, PC-3/M, was isolated from a PC-3-induced mouse tumor. Karyotypic analysis of PC-3 by Q- and C-banding showed the cells to be aneuploid at all culture passage levels. The modal chromosome number shifted from 62 to 55 between the 5th and 50th passages. PC-3 has a unique karyotype. Chromosomes 2, 3, 5, 15, and Y were always absent. At least 11 different marker chromosomes were observed. The subline, PC-3/M, had a similar karyotype and retained the parental PC-3 markers. PC-3/M had a more restricted chromosomal frequency distribution range. Nearly 73% of the PC-3/M cells examined had 60 or 61 chromosomes in contrast to the wide distribution seen in PC-3. Silver staining for nucleolus organizer regions indicated that the number of functional nucleolus organizer regions in PC-3 was proportional to the number of acrocentric chromosomes. Banding analysis of PC-5-PI isolated from primary prostatic adenocarcinoma indicated that this line also had a characteristic karyotype with 28% pseudodiploid and 72% pseudotetraploid components. All metaphases examined were partially trisomic in chromosome 9 and lacked a demonstrable Y chromosome.

One-year follow-up of cognitive-behavioral therapy for obese individuals with binge eating disorder.

The results of a 1-year posttreatment follow-up of 93 obese women diagnosed as having binge eating disorder (BED) and treated with group cognitive-behavioral therapy (CBT) followed by weight loss treatment are described. The group as a whole maintained both reductions in binge eating and abstinence rates fairly well. However, they regained the weight lost during treatment. Those who stopped binge eating during CBT maintained a weight loss of 4.0 kg over the follow-up period. In contrast, those who continued to binge gained 3.6 kg. Twenty-six percent of those abstinent after CBT met criteria for BED at follow-up and had gained weight, whereas the remaining 74% had lost weight. Stopping binge eating appears critical to sustained weight loss in BED.

Does interpersonal therapy help patients with binge eating disorder who fail to respond to cognitive-behavioral therapy?

The aim of this quasi-experimental study was to examine the effectiveness of group interpersonal therapy (IPT) in treating overweight patients with binge eating disorder who did not stop binge eating after 12 weeks of group cognitive-behavioral therapy (CBT). Participants in this study were randomly allocated to either group CBT or to an assessment-only control group. After 12 weeks of treatment with CBT, 55% of participants met criteria for improvement and began 12 weeks of weight loss therapy, whereas the nonresponders began 12 weeks of group IPT. Over the 24-week period, participants who received treatment reduced binge eating and weight significantly more than the waiting-list control group. However, IPT led to no further improvement for those who did not improve with CBT. Predictors of poor outcome were early onset of, and more severe, binge eating.

Maintenance following a very-low-calorie diet.

The authors posed 2 questions in this randomized study of maintenance procedures in which participants were followed for 15 months after completion of a very-low-calorie diet: Would stimulus narrowing during the reintroduction of solid food, achieved by the use of prepackaged foods, improve weight losses and the maintenance of those losses as compared with the use of regular food? Would reintroduction of foods dependent on progress in losing or maintaining weight be superior to reintroduction on a time-dependent basis? Neither the stimulus narrowing condition nor the reintroduction procedure enhanced either maximum weight loss or maintenance of those losses. The stimulus narrowing condition appeared to be poorly tolerated; compliance and attendance were poorer in this condition than in the regular food condition.

A thermosensitive lesion in a Chinese hamster cell mutant causing differential effects on the acidification of endosomes and lysosomes.

We previously described the isolation and preliminary characterization of a Chinese hamster ovary cell mutant, termed G.7.1, that carried a temperature-sensitive, conditional-lethal lesion affecting the acidification of vesicles in crude cellular extracts (Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D. K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907-1916). In the present report, we have separated lysosomal vesicles from more buoyant nonlysosomal vesicles by centrifuging cell extracts with Percoll and correlated the acidification defect with nonlysosomal vesicles, including endosomes, but not with secondary lysosomes. Moreover, the acidification of nonlysosomal vesicles prepared from mutant cells grown at the permissive temperature was more sensitive to thermal inactivation than similar vesicles from parental cells, implying that a heat-sensitive component is a normal resident of nonlysosomal vesicles in the mutant. This heat-sensitive component is apparently not associated with lysosomes, or if it is, it does not inhibit lysosomal acidification at the nonpermissive temperature. We also found that the transferrin-mediated uptake of iron is inhibited by 50% in the mutant cells at the nonpermissive temperature and that the inhibition cannot be accounted for by reduced binding or internalization of transferrin.

Thermolabile proton translocating ATPase and pump activities in a clathrin-coated vesicle fraction from an acidification defective Chinese hamster cell line.

We have recently described a mutant of Chinese hamster ovary cells, termed G.7.1, that contains a temperature-sensitive, conditionally lethal mutation resulting in defective vacuolar acidification (Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D.K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907-1916). To further characterize the lesion, clathrin-coated vesicles were partially purified from wild type and G.7.1 cells, and the thermolabilities of vanadate and oligomycin-insensitive, N-ethylmaleimide-sensitive, H+-ATPase activity, 32Pi-ATPase exchange activity, and proton pumping were compared. All three parameters of H+ pump activity were markedly diminished by preincubation at 44 degrees C for vesicles harvested from the G.7.1 cells, but not for those from wild type cells. Phosphatidylserine did not protect against heat inactivation in vesicle fractions prepared from G.7.1 cells. The results suggest that the mutation responsible for defective acidification in G.7.1 cells is expressed at the level of the proton pump of organelles present in our clathrin-coated vesicle-enriched preparation.

Evidence for penetration of diphtheria toxin to the cytosol through a prelysosomal membrane.

To kill mammalian cells, diphtheria toxin must be endocytosed and encounter a low pH within intracellular vesicles. The low pH initiates penetration of the catalytically active A fragment of the toxin through a membrane and into the cytosol where the A fragment arrests protein synthesis. To investigate whether penetration occurred through a prelysosomal or a lysosomal membrane, we studied the effect of low temperature on the entry of the toxin into the cytosolic and lysosomal compartments. The toxin arrested protein synthesis at 15 degrees C, indicating entry into the cytosol; however, access to lysosomes was apparently blocked at 15 degrees C, suggesting that the toxin had encountered a low pH before reaching lysosomes and had penetrated a prelysosomal membrane. To further investigate the possibility of prelysosomal acidification, we measured the time required for the toxin to encounter a low pH after endocytosis. Acidification occurred within 3 to 4 min after the toxin was internalized into vesicles. This interval is consistent with prelysosomal acidification since the entry of endocytosed ligands into secondary lysosomes usually takes more than 3 to 4 min.

The effects of extending cognitive-behavioral therapy for binge eating disorder among initial treatment nonresponders.

OBJECTIVE: The study was designed with the aim of determining whether extending group cognitive-behavioral therapy (CBT) would enhance outcome among individuals with binge eating disorder (BED) who failed to stop binge eating after an initial 12-week CBT intervention. METHOD: Forty-six participants who met diagnostic criteria for BED were randomly allocated to either a 12-week group CBT intervention or a waiting list control condition. At the end of 12 weeks, treated participants who met clinical criteria for improvement subsequently received 12 sessions of behavioral weight loss. Remaining participants received 12 additional sessions of CBT for binge eating. RESULTS: Fifty percent of treated participants improved with the initial 12-week course of CBT. There was a strong trend for the extension of CBT to affect improvement in binge eating among initial nonresponders (6 of 14 subjects no longer met diagnostic criteria for BED). Overall, extending CBT led to clinical improvement in 66.7% of all treated participants, with treatment gains occurring through session 20. DISCUSSION: The results suggest that an extended course of CBT (i.e., longer than 12 weeks) will likely maximize the number of potential responders to treatment.