A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.74.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping. There have been conflicting reports and theories about the pH inside lysosomes. This controversy has been the subject of a review by Tager and Reijngoud (1). Isolated lysosomes have an internal pH about 1 unit lower than that of the medium, apparently as a consequence of a Donnan equilibrium (2-4). However, there is some evidence of an energy-dependent mechanism that is capable of lowering the pH (5, 6). In living cells, various weakly basic substances are concentrated in lysosomes as a consequence of lysosomal acidity (7). When the intralysosomal concentration of these substances becomes sufficiently high, the lysosomes swell osmotically to form large vacuoles (8, 9). Vacuoles of similar appearance have been observed in cells exposed to the acidic ionophore X537A (10).Attempts have been made to estimate the pH inside lysosomes by visual inspection of color changes in pH indicator dyes (see ref. 1). Here we describe a quantitative method for the measurement of intralysosomal pH based on the pH-dependent fluorescence signals from fluorescein isothiocyanate-labeled dextran (FD) in the lysosomes of living cells. Control experiments indicate that these signals should provide an accurate measure of pH. We report the results of some measurements of pH in the lysosomes of living cells under various conditions that support the existence of an active process of pH maintenance and provide some confirmation of the theory of pHdependent concentration of certain substances in lysosomes (7). MATERIALS AND METHODSMouse peritoneal macrophages isolated by the method of Cohn and Benson (11) were cultured in modified Eagle's medium (12) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fa...

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The spectral characteristics of dextran, labeled with fluorescein, depend upon pH . We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions . The pH of the medium has no effect on the intralysosomal pH . Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH . However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form . Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH . This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A.The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.In a previous paper (6) we reported the results of experiments showing that exposure of macrophages to medium containing any one of a large number of weak bases results in the uptake of the bases into lysosomes, and, when the concentration in lysosomes becomes sufficiently high, osmotic swelling of these particles to form large phase-lucent vacuoles occurs. We showed that the concentration of the free base in the medium is the determining factor for uptake and vacuolation, and that the protonated base in the medium has no effect. Widely different concentrations are required to elicit vacuolation by different bases.We have described previously a fluorescence probe technique for the measurement of intralysosomal pH (5) . In this study we use this technique to explore, in some detail, the pH changes caused in the lysosomes by some of these weak bases, and we discuss the results in terms of the leakage of protons out of lysosomes in the form of protonated base. We conclude that the high acidity in lysosomes is the consequence of an active process of proton secretion into lysosomes . MATERIALS AND METHODS Cell CultureOur culture procedures for mouse peritoneal macrophages, adapted from This paper is appearing without fmal revision due to the untimely death of Dr . Brian Poole.THE JOURNAL OF CELL BIOLOGY " VOLUME 90 SEPTEMBER 1981 665-669 © The Rockefeller University Press " 0021-9525/81/09/0665/05 $1 .00Cohn and Benson (2), have been described fully in the previous paper in this series (6). Lysosomal pH MeasurementThis technique has been described in more detail elsewhere (5) . Briefly, cells were exposed to medium containing 1 mg/ml fluorescein-labeled dextran for 24 h. They were then washed free of medium and the cover slips were mounted in a special holder in a regular fluorescence cell that was perfused with various media . The fluorescence of the cells was measured with a Hitachi Perkin-Elmer MPF...

The COVID-19 pandemic continues to ravage the world, with the United States being highly affected. A vaccine provides the best hope for a permanent solution to controlling the pandemic. However, to be effective, a vaccine must be accepted and used by a large majority of the population. The aim of this study was to understand the attitudes towards and obstacles facing vaccination with a potential COVID-19 vaccine. To measure these attitudes a survey was administered to 316 respondents across the United States by a survey corporation. Structural equation modeling was used to analyze the relationships of several factors with attitudes toward potential COVID-19 vaccination. Prior vaccine usage and attitudes predicted attitudes towards COVID-19 vaccination. Assessment of the severity of COVID-19 for the United States was also predictive. Approximately 68% of all respondents were supportive of being vaccinated for COVID-19, but side effects, efficacy and length of testing remained concerns. Longer testing, increased efficacy and development in the United States were significantly associated with increased vaccine acceptance. Messages promoting COVID-19 vaccination should seek to alleviate the concerns of those who are already vaccine-hesitant. Messaging directed at the benefits of vaccination for the United States as a country would address the second predictive factor. Enough time should be taken to allay concerns about both short- and long-term side effects before a vaccine is released.

Acute renal failure (ARF), characterized by sudden loss of the ability of the kidneys to excrete wastes, concentrate urine, conserve electrolytes, and maintain fluid balance, is a frequent clinical problem, particularly in the intensive care unit, where it is associated with a mortality of between 50% and 80%. In this review, the epidemiology and pathophysiology of ARF are discussed, including the vascular, tubular, and inflammatory perturbations. The clinical evaluation of ARF and implications for potential future therapies to decrease the high mortality are described.

Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-a-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.

With few exceptions, weakly basic compounds that are sufficiently lipophilic in their neutral forms and sufficiently hydrophilic in their protonated forms accumulate in lysosomes. When the concentration within the lysosomes becomes sufficiently high, osmotic swelling occurs . The cells then take on a vacuolated appearance . The concentrations at which different weak bases cause lysosomal vacuolation vary over almost three orders of magnitude. For any particular weak base, it is the concentration of the neutral form that determines the extent of uptake and the degree of vacuolation. Chloroquine is anomalous in that concentrations > -30 [LM cause less uptake and less vacuolation than do lower concentrations .It has been found that the treatment of cells with a variety of chemical compounds leads to the formation, in the cytoplasm, ofmanylarge vacuoles (3,20,22,23). Many ofthese substances, the best know being neutral red, are weak bases. De Duve et al. (7) have proposed a quantitative theory to account for the accumulation, in lysosomes, of weakly basic substances and the formation of vacuoles. This theory is the logical consequence of three assumptions. First, that the plasma and lysosomal membranes are highly permeable to the neutral forms of weak bases. Second, that these same membranes are impermeable, or very slightly permeable, to the protonated forms ofthe weak bases. And third, that the pH inside the lysosomes is considerably lower than it is outside the lysosomes . These assumptions have the following consequences. First, that weak bases will be trapped by protonation inside lysosomes and accumulate there. And, second, that when the concentration of the base inside the lysosomes approaches isotonicity, water will enter osmotically and the lysosomes will swell to form large vacuoles.At the time this theory was proposed we had no quantitative measure of the pH in lysosomes. Recently we have devised a technique to measure this parameter in mouse peritoneal macrophages (13) and have discovered that the pH rises in the presence of weak bases, a possibility not considered in the original theory (7) .In this paper we will examine, in some detail, the ability of This paper is appearing without final revision due to the untimely death of Dr . Brian Poole .a number of weak bases to induce vacuolation in mouse peritoneal macrophages and the kinetics of uptake of these bases into the cells. In subsequent papers we will explore, in more detail, the pH changes that occur in the lysosomes of mouse peritoneal macrophages exposed to weak bases, and the effect of these compounds on lysosomal protein degradation (18). MATERIALS AND METHODS Cell CultureMouse peritoneal macrophages were isolated by the method of Cohn and Benson (6) from NCS strain mice . They were cultured in Dulbecco's modified Eagle's minimum essential medium (l2) at pH 7.6 (unless otherwise indicated), containing 20% fetal calf serum, 25 Pg/ml Gentamicin, and 2.5 jug/ml Fungizone in glass Leighton tubes, either on the tube surface or on cover slip...

The degradation of cellular proteins in fibroblasts, both those of rapid and those of slow turnover rates, was inhibited by low concentrations of chloroquine or neutral red in the medium. Cells inhibited by chloroquine can be inhibited further by fluoride. Chloroquine was taken up by the fibroblasts and the concentration in the cells reached several hundred times that in the medium. Isopycnic fractionation studies showed that within the cells the chloroquine was concentrated in the lysosomes, and that these chloroquine-containing lysosomes had a lower equilibrium density than the lysosomes of untreated cells. Chloroquine, at concentrations attained inside the lysosomes, inhibited cathepsin Ba but not cathepsin D. It is concluded that chloroquine impairs the breakdown of cellular proteins after these have entered the lysosome system, probably through inhibition of cathepsin Ba.Very little is known about the mechanism of degradation of cellular protein during normal protein turnover. It has long been established that the process requires an energy supply (21) and cellular integrity (20). The only known intracellular proteolytic system capable of degrading proteins to their constituent amino acids is in the lysosomes (8). It is well established that some cellular proteins are degraded in lysosomes after the formation of autophagic vacuoles, but the extent to which this process contributes to normal protein turnover is unknown.The previous paper in this series (18) showed that the degradation of proteins with long half-lives was inhibited in rat fibroblasts by a factor in fresh medium, while the degradation of proteins with short half-lives was unaffected. The degradation of both classes of protein was inhibited by fluoride through some mechanism more specific than the inhibition of glycolysis. In this paper we report the effects of chloroquine. MATERIALS AND METHODSFibroblasts, isolated from trypsinized rat embryos, were cultivated in basal Eagle's medium in culture flasks (75 cm 2 growth surface, 30 ml medium) or in Leighton tubes (4.8 cm 2 growth surface, 2 ml medium) and used for experiments between the third and the fifth passage, after confluency was reached. In experiments with labeled media the cells were washed four times with Hanks' solution at each change of medium. At the end of the experiment the washed cells were dissolved in 0.1 M NaOH-0.4% deoxycholate. Total and 8% trichloroacetic acid-soluble radioactivity was measured on each sample of cells and medium as described previously (18). Radio- 430