As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification.Vacuoles were labeled with 6-carboxyfluorescein with the membrane-permeant precursor 6-carboxyfluorescein diacetate. Dual-excitation flow cytometry was used to calibrate the pH-dependence of 6-carboxyfluorescein fluorescence in vivo.Vacuoles in wild-type yeast were mildly acidic, pH 6.2, in cells grown under several different conditions. Cultures labeled with 6-carboxyfluorescein were screened by fluorescence-ratio microscopy to detect mutants that had defects related to vacuolar acidification. A recessive nuclear mutation, vphl-1, caused an abnormally high vacuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine. Acidification in a pepl2::LEU2 mutant appeared defective by fluorescence-ratio microscopy and qulnacrineuptake assays, but the vacuolar pH in thepepl2::LEU2 mutant was nearly normal (pH 6.3) in flow cytometric assays.Vacuoles in Saccharomyces cerevisiae are analogous to lysosomes of other organisms. Both types of organelles contain similar sets of proteases and other hydrolytic enzymes (1, 2), and both are energized and acidified by similar membrane-bound, proton-translocating ATPases (H+-ATPase) (3-6). Vacuolar proteases are required for intracellular protein turnover induced by nitrogen starvation (7). Apart from that typically "lysosomal" activity, a variety of additional functions have been ascribed to the vacuole, including maintenance of cytosolic amino acid homeostasis (8, 9), regulation of cellular calcium metabolism (8, 10), and others (11). Genetic analysis of protease-deficient mutants has been instrumental in delimiting the physiological significance of the vacuolar proteases (7 METHODSStrains and Culture Conditions. The wild-type strain was the diploid formed by mating strains X2180-1A and X2180-1B. Mutant strains and genotypes were BJ4895, a/a vphl-J/ vphl-1 trpl/+ leu2/+ +/ura3-52 and BJ4984, a/a pepl2:: LEU2/pepl2::LEU2 ura3-52/ura3-52 leu2-1/leu2-1 hisl1+ ade6/+. Growth media YPD and SC have been described (12). Cells in the logarithmic-growth phase were prepared by overnight growth in YPD or SC (10 ml) at 30'C in roller tube cultures. Culture densities were maintained below 2 x 107 cell per ml by dilution with YPD or SC as required.Labeling with Fluorescent Dyes. Medium for labeling with 6-CF diacetate (6-CFDA; C1362; Molecular Probes) was prepared by adding 50 mM citric acid to YPD, adjusting the pH to 3.0 with 12 M HCl, autoclaving, and then filtering (0.2 gm membrane filter); 6-CFDA was added to the medium immediately before use by dilution from 5 mM stock solutions in dimethyl sulfoxide; unless otherwise noted, the final 6-CFDA concentration was 5 1LM. Logarithmic-phase cells were harvested by membrane filtration, resuspended at 2 X 107 cell per ml in the labeling medium, and incubated with shakin...

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To produce concentrations of zidovudine (AZT) in plasma and cerebrospinal fluid that would provide constant inhibition of the replication of human immunodeficiency virus (HIV), we gave AZT by continuous intravenous infusion to 21 children ranging in age from 14 months to 12 years who had acquired HIV infection through transfusions or perinatally. All patients were symptomatic before AZT treatment (Class P2 of the Centers for Disease Control); 13 (62 percent) had evidence of neurodevelopmental abnormalities. The mean CD4/CD8 ratio was 0.18; 11 patients had CD4 counts below 0.2 x 10(9) per liter. We administered AZT at four dose levels: 0.5, 0.9, 1.4, and 1.8 mg per kilogram of body weight per hour. The plasma drug concentrations achieved at the respective dose levels were 1.9 +/- 0.3, 2.8 +/- 1.4, 3.1 +/- 1.1, and 4.5 +/- 1.0 microM. The steady-state cerebrospinal fluid:plasma ratio was 0.24 +/- 0.07. The only evidence of toxicity was bone marrow suppression. Transfusion was required in 14 patients because of low levels of hemoglobin (5 mmol per liter [less than 8 g per deciliter]). Dose-limiting neutropenia (less than 0.5 x 10(9) polymorphonuclear leukocytes per cubic millimeter) occurred in most patients who received doses of 1.4 mg per kilogram per hour or more. Improvement in neurodevelopmental abnormalities occurred in all 13 children who had presented with encephalopathy before treatment. Serial measurements of IQ before therapy and after three and six months of continuous therapy with AZT showed that IQ scores, including those for verbal and performance IQ, rose in these 13 patients and in 5 other children who had no detectable evidence of encephalopathy before treatment. Most patients also had increased appetite and weight, decreased lymphadenopathy and hepatosplenomegaly, decreased immunoglobulin levels, and increased numbers of CD4 cells. In some patients the improvement in the features of encephalopathy occurred despite the absence of immunologic improvement. We conclude that AZT is beneficial in children with symptomatic HIV infection, especially those with encephalopathy (which may be subclinical), and that the optimal continuous intravenous dose of AZT in children is between 0.9 and 1.4 mg per kilogram per hour.

Abstract. Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants.After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 ~tg/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 106 cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution. SEVERAL lines of evidence suggest that uptake of the small molecule, Lucifer yellow (Riezman, 1985), and macromolecules (Makarow, 1985;Makarow and Nevalainen, 1987) can occur in the yeast Saccharomyces cerevisiae by a mechanism that resembles fluid-phase endocytosis. In a potentially related development, recent reports strongly support a role for receptor-mediated endocytosis in the yeast cell's response to a-factor mating pheromone (Jenness and Spatrick, 1986;Chvatchko et al., 1986). Fluidphase endocytosis should provide a port-of-entry for loading cells with a variety of otherwise impermeant metabolites or tracer molecules. Its occurrence in yeast encourages the application of the powerful genetics of this organism not only for the analysis of endocytotic mechanisms but also for the analysis of phenomena dependent on internalization of impermeant substances.In the latter of these applications, we have attempted to replicate the reported labeling of yeast vacuoles by high molecular weight, FITC-conjugated dextran (FD) t to investigate problems in organelle biogenesis and function (Makarow, 1985;Makarow and Nevalainen, 1987). As will be reported here, we found that the fluorescent staining of yeast vacuoles by "70-kD FD" results from the uptake of low molecular weight impurities in commercial samples of FD. FITC appears to be the most potent of these impurities, and its uptake most likely reflects simple diffusion and trapping rather than endocytosis.Our results suggest that attempts to introduce macromolecular materials into whole yeast cells by an endocytotic route may be ill-advised. Also, in view of the potential reactivity of FITC (particularly if concen...

Abstract. To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic acidification, as measured by transferrin acidification, is normal in this cell line. However, a difference in subsequent endocytic acidification was observed in CHL60-64. In the parental cells, internalized dextran was fully acidified to approximately pH 5.5 within 1 h. In CHL60-64, the pH in the endocytic compartment was only 6.1 after 1 h and remained as high as 5.8 for at least 4 h. After an 8-h incubation, the pH decreased to 5.5, indicating that the second phase of acidification is only slowed in CHL60-64, and not blocked. Consistent with this retarded acidification, ATP-dependent acidification in vitro (as measured by acridine orange accumulation) was reduced in both the lysosomal fraction and the endosomal fraction isolated from CHL60-64. A decrease in the in vivo rate of acridine orange accumulation after perturbation with amine was also observed. In addition to amine resistance and defective acidification, CHL60-64 was found to be resistant to vacuolation in the presence of chloroquine and ammonium chloride, and was resistant to ouabain. Further studies on this new class of endocytosis mutant, in combination with existing mutants, should help to clarify the mechanisms responsible for the regulation of endocytic acidification. M^Nv viruses and toxins require internalization and exposure to an acidic pH in order to penetrate into the cytosol (8,20,24). A number of cell lines that are defective in endocytosis and/or endocytic acidification have been isolated by taking advantage of this fact. Robbins et al. (6,15,16,19) have described a number of cell lines, selected for diphtheria toxin resistance and impaired mannose-6-phosphate receptor activity, that are resistant to infection by Sindbis virus and vesicular stomatitis virus, as well as protein toxins. Some mutants were found to be defective in the uptake of iron from transferrin, a phenotype linked to their defect in vacuolar acidification, and some exhibited altered Golgi apparatus functions as well. Selection for resistance to both modeccin and diphtheria toxin enabled Draper and co-workers (7, 22) to isolate Chinese hamster ovary cell lines that were temperature sensitive for viability, resistance to protein toxins (diphtheria toxin, modeccin, Pseudomonas exotoxin A), and ATP-stimulated acidification of endocytic vesicles. The temperature-sensitive lesion in viability could be overcome by the addition of FeSO4 to the growth media. Merion et al. (9) have characterized Chinese hamster ovary acidification mutants resistant to Pseudomonas exotoxin A, which were found to be cross-resistant to diphtheria toxin and several animal viruses. Monensin-resistant mutants have been isolated th...

Prostate cancer can have diverse effects on patients' quality of life (QoL). Standard QoL questionnaires do not address all of the concerns expressed by such patients. The primary purpose of this study was to identify those issues with the greatest influence on the QoL of patients with prostate cancer. A secondary aim was to compare the performance of the Schedule for the Evaluation of Individual Quality of Life-Direct Weighting (SEIQoL-DW) semi-structured interview with the Functional Assessment of Cancer Therapy-Prostate questionnaire (FACT-P). A mixed population of patients with prostate cancer (including those with localized and metastatic disease) completed the SEIQoL-DW and the FACT-P. The SEIQoL-DW was satisfactorily completed by 180 patients, including 93 patients with metastatic disease. Patients identified 144 separate QoL concerns, and these were then independently grouped by three of the authors into 13 distinct themes. The most frequently identified themes were 'leisure and hobbies', 'family' and 'health'. The themes that patients considered to be the most important were 'partner/spouse', 'family' and 'health'. Patients were most satisfied with their QoL in the domains of 'family', 'partner/spouse' and 'friends'. They were least satisfied with 'sexuality', 'mobility' and 'psychological factors'. Patients with metastatic disease rated their QoL significantly (Po0.0001) lower than other patients using the FACT-P, but not using the SEIQoL-DW (P ¼ 0.07). Patients with prostate cancer identified numerous QoL concerns that are not included (or are underrepresented) in standard health-related QoL questionnaires such as the FACT-P. Health-related QoL questionnaires may underestimate the QoL of patients with metastatic disease.

Summary Three human breast cancer cell lines ZR-75-1, MDA-MB-436 and MCF-7 were found to contain respectively, 3.06, 2.69 and 1.86fmol of somatostatin-like immunoreactivity (SLI) per 106 cells. Since SLI is undetectable in the passaging media it must, therefore, be synthesised by the cells. In the presence of fetal calf serum the cells were growth inhibited by addition of somatostatin or its long-lasting analogue, Sandostatin, but only after 3 days of continuous exposure. A 1-day exposure to either peptide had little or no effect on subsequent cell growth in peptide-free medium. Inhibition of cell proliferation is not due to cytotoxic effects of the dose used (500ngml-1, each) since both peptides caused short-term stimulation of growth in the absence of serum.