Characterization of Hela cell vacuoles induced by Helicobacter pylori broth culture supernatant

Characterization of HeLa Cell Vacuoles Induced by Hekobacter py/ori Broth Culture Supernatant TIMOTHY L. COVER, MD, SUSAN A. HALTER, AND MARTIN J. BLASER, MD Helicobaeter pylori broth culture supematants induce eukaryotic cell vacuolation in vitro, a phenomenon

that has been attributed to cy-

totoxic activity. We sought to characterize further the vacuolation of HeLa cells that occurs in response to Hpylori

culture supematant.

Nascent vacuoles were detectable by electron microscopy

after 90

minutes of incubation with H pylon’ supernatant and were not associated with any identifiable

organelle. After 6 days of incubation

with H pylon’ supernatant, vacuoles were membrane-bound tures filled with electron-dense

debris, which resembled

struc-

secondary

lysosomes. Acid phosphatase

activity was detected within the vac-

uoles. The vacuoles

by H pylori

compared

induced

with vacuoles induced

supernatant

by trimethylamine,

were then

a weak base

known to induce lysosomal swelling. Neutral red dye rapidly entered the vacuoles induced

by either H pylon’ supematant

or trimethyl-

amine, and both types of vacuoles were reversible. Compared trimethylamine-induced

with

vacuoles, the vacuoles induced by Hpylori

supernatant were larger and typically lacked a limiting membrane.

MD,

igenic Hpylori strains is more prevalent among Hpyloriinfected persons with duodenal ulceration than among infected patients with gastritis alone; hence, cytotoxin production may be an important virulence factor5r6 The vacuolating cytotoxic activity in H pylori culture supernatants is abolished by heatin to greater than 70°C or by incubation with proteases, 89 which suggests that the phenomenon is mediated by a protein. Hpylori cytotoxic activity is distinct from urease, and a serum neutralizing antibody response to the cytotoxin occurs in H pyloriinfected persons.“,” The mechanism whereby H pylori cytotoxin induces cell vacuolation has not yet been determined. Therefore, the goal of this study was to characterize further the vacuolation of HeLa cells that occurs in response to cytotoxic H pylori supernatant.

In the early stages of formation, vacuoles induced by trimethylamine were labeled

by lucifer yellow, a pinocytotic

pyloricytotoxin-induced trimethylamine-induced compartments,

marker, whereas

H

vacuoles were not. These data suggest that vacuoles

arise

directly

from

endocytic

whereas H pylon’ cytotoxin induces vacuole forma-

tion via an autophagic mechanism. HUM PATHOL 23: 1004- 10 10. This is a US government

work. There are no restrictions on its use.

A large body of evidence indicates that Helicobacter is the cause of chronic active gastritis in humans.’ In addition, there is a strong association between H pylori infection and peptic ulcer disease.2.3 H pylori rarely invades the gastric mucosal epithelium4; therefore, secreted products are likely to play a role in the pathogenesis of gastric inflammation and gastric epithelial ‘damage. Cell-free broth culture supernatants from approximately 50% of H pylori strains induce eukaryotic cell vacuolation in vitro, a phenomenon that has been attributed to cytotoxic activity.5-s Infection with cytotox-

pylori infection

From the Infectious Diseases and Laboratory Service Sections, Department of Veterans Affairs Medical Center, and Departments of Medicine, Microbiology, and Pathology, Vanderbilt University School of Medicine, Nashville, TN. Accepted for publication October 25, 1991. Dr Cover is a recipient of a Searle Scholars Fellowship Award from the Infectious Disease Society of America. Supported in part by a grant from The Procter & Gamble Company. Key words: Helicobacter pylori, cytotoxin, vacuole, autophagy. Address correspondence to Timothy L. Cover, MD, Division of Infectious Diseases, A-3310 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2605. This is a US government work. There are no restrictions on its use. 0046-8177/92/2309-0006$0.00/0

1004

METHODS Bacterial Strains and Toxin Production H pylori strains 60 190 and TxJOa, tax+ and tax-, tively,7,8 were cultured in Brucella broth containing

respec-

5% fetal bovine serum for 48 hours on a rotary shaker at 37°C in ambient air supplemented with 5% COP, and the cultures were then centrifuged at 10,000 X g for 20 minutes.’ The supernatant was concentrated 30-fold with a lOO-kd cutoff ultrafiltration membrane, and the retentate was sterilized by passage through a 0.2~pm filter.’ When tested in cell culture as previously described,7 a 1:160 dilution of concentrated supernatant from H pylori strain 60190 produced vacuolation of HeLa cells, whereas a 1: 10 dilution of supernatant from H pylori strain Tx30a failed to produce cell vacuolation. Previous studies have demonstrated that the urease activities and ammonia concentrations in culture supernatants from H pylon’ 60190 and Tx3Oa are not significantly different.g

Induction of Vacuole Formation in HeLa Cells HeLa cells were cultured in modified Eagle’s minimal essential medium (Flow, Irvine, CA) containing 10% fetal bovine serum (MEM-FBS) at 37°C in ambient air supplemented with 5% CO,. In preparation for electron microscopic examination, cells were seeded into 25-cm” tissue culture flasks (lOfi cells per flask) and allowed to adhere for 24 hours. The overlying medium was then removed and cells were incubated for the specified times at 37°C with 5 mL of fresh MEM-FBS containing concentrated Hpykn-i culture supernatant or 25 mmol/ L trimethylamine (Sigma, St Louis, MO).

Preparation

of Cells for Electron Microscopy

After incubation with test substances cells were detached with trypsin-EDTA (Gibco, Grand Island, NY) and resuspended

H MOW CYTOTOXIN-INDUCED

in MEM-FBS. The suspension was centrifuged at 3,000 X g for 15 minutes, and the cell pellet was fixed overnight at 4’C in 0.1 mmol/L phosphate buffered saline (pH 7.4) containing 3% glutaraldehyde. The cells were postfixed in osmium tetroxide, dehydrated in graded alcohols, en bloc stained with

uranyl acetate, and embedded in Spurr (Electron Microscopy Sciences, Fort Washington, PA).” Thick sections were cut and stained with toluidine blue. Thin sections were mounted on copper grids and stained with lead citrate. The sections were viewed with a Hitachi H60 electron microscope.

Acid Phosphatase

Staining

HeLa cells were stained using a modification of the Gomori acid phosphatase staining method.‘3-‘5 After incubation with MEM-FBS containing Hpylori supernatant or MEM-FBS alone, cells were harvested as described above and fixed for 30 minutes at 4°C in 0.1 mol/L sodium cacodylate (pH 7.4) containing 1% glutaraldehyde and 1% paraformaldehyde. The cell pellet was rinsed with 0.1 mol/L sodium cacodylate containing 7% sucrose, stored overnight at 4”C, and then incubated for 30 minutes at 37°C in 50 mmol/L sodium acetate (pH 5.0) containing 10 mmol/L disodium @-glycerophosphate (Sigma) and 3.3 mmol/L lead nitrate. The cells were rinsed with sodium cacodylate containing 7% sucrose, fixed for 60 minutes at 4°C in sodium cacodylate containing 1% osmium tetroxide, and then dehydrated, embedded, cut, and stained as described above.

Neutral Red Staining After incubation with test substances for 18 hours, cells were stained with 0.05% neutral red in MEM-FBS for 4 minutes and washed with 0.9% NaCl. Intracellular neutral red was quantitated spectrophotometrically, as previously described.g

Labeling of Endocytic With Lucifer Yellow

Compartments

To label cellular endocytic compartments, adherent HeLa cells were incubated with 100 pg/mL lucifer yellow CH (lithium salt, Sigma) in MEM-FBS in the presence or absence of test substances.16 After washing with 0.9% NaCl, unfixed live cells were examined using an Olympus BHS microscope with a BH2RFL fluorescent attachment.

Statistical

Methods

Experiments with neutral red or lucifer yellow uptake were performed in triplicate, and results are expressed as the mean f SEM. Analysis of optical density values was performed using Student’s t-test.

RESULTS Characterization

of Vacuole Formation

To study ultrastructural characteristics during the process of vacuole formation,

of vacuoles

HeLa cells were incubated with MEM-FBS containing a 1:20 dilution of concentrated supernatant from Hpylori strain

60190 for time intervals ranging from 30 minutes to 6 days. HeLa cells incubated for 18 hours with MEM-FBS alone contained essentially no vacuoles (Fig 1A); similarly, concentrated supernatant from the tox- Hpylori strain Tx3Oa or concentrated, uninoculated bacterial culture medium failed to induce cell vacuolation (not shown). Vacuoles were detectable after 90 minutes of

1005

VACUOLATION

(Cover et al)

incubation with supernatant from H pylori strain 60 190; structures resembling secondary lysosomes were not increased in size (Fig 1B). At 4 hours (Fig 1C) most vacuoles were discrete structures that contained electrondense material. No alteration in the structure of mitochondria or endoplasmic reticulum was observed. By 18 hours (Fig 1D) coalescent vacuoles filled the cytoplasm and displaced the nucleus to one side in many cells. After 6 days of incubation (Fig 1 E) viable cells contained vacuoles filled with electron-dense debris; some of the vacuoles were limited by membranes. Nonviable vacuolated cells were also observed after 6 days of incubation (not shown). These observations indicated that in the earliest stages of formation, vacuoles were not associated with or derived from any clearly identified organelle. Acid Phosphatase

Staining of Cell Vacuoles

The presence of electron-dense debris in vacuoles suggested the occurrence of a degradative process and, hence, cells were stained for acid phosphatase activity, a lysosomal enzyme marker.” After 6 hours of incubation with Hpylori supernatant electron-dense reaction product was identified in some, but not all, HeLa cell vacuoles (Fig 2). H pylori supernatant-treated cells stained with a reaction mixture lacking sodium glycerophosphate or containing sodium fluoride did not contain reaction product. In control cells incubated with MEMFBS alone, reaction product was present only in rare, small, discrete structures consistent with normal lysosomes (not shown). These studies suggested that Hpylori supernatant-induced vacuoles contained lysosomal enzymes. Ultrastructural Comparison Induced by Trimethylamine Supernatant

of Cell Vacuoles or H pylori

Vacuoles resembling those produced by H pylori supernatant have been induced previously by weak bases.18W20Therefore, the ultrastructure of cell vacuoles induced by Hpylori supernatant was compared with that of vacuoles induced by trimethylamine, a lysosomotropic weak base.” HeLa cells were incubated for 24 hours with MEM-FBS containing 25 mmol/L trimethylamine and were examined by electron microscopy (Fig 3). Most of the cells contained abundant cytoplasmic vacuoles, many of which were limited by membranes. In comparison to the vacuoles induced by H pylon supernatant, trimethylamine-induced vacuoles appeared smaller and more regular in shape, and more frequently contained a limiting membrane. Further studies were then performed to compare the functional properties of the vacuoles induced by these two agents. Incorporation

of Neutral Red into Vacuoles

To compare the intravacuolar pH of H pylori supernatantand trimethylamine-induced vacuoles, we studied the entry of neutral red dye into cell vacuoles.g HeLa cells were incubated with test substances for 18 hours and were then stained for 4 minutes with 0.05% neutral red. Neutral red avidly stained the vacuoles in-

HUMAN PATHOLOGY

Volume 23, No. 9 (September

1992)

FIGURE 1. Electron micrographs of HeLa cells. (A) A cell incubated for 18 hours in MEM-FBS alone is shown. Abundant mitochondria are present in the cytoplasm, but no vacuoles are present. The arrowhead denotes a structure consistent with a secondary lysosome. (Bar, 1 pm.) (B) A cell incubated with supernatant from H pylori strain 60190 for 90 minutes is shown. Several cytoplasmic vacuoles with irregular borders and without lining membranes have formed. Myelin tigures are visible within two of the vacuoles (arrows). The nucleus, mitochondria. and endoplasmic reticulum are unchanged in appearance. Structures resembling secondary lysosomes (double curved arrow) are not increased in size compared with panel A. (Bar, 1 pm.) (C) After 4 hours of incubation with H pylori supernatant, numerous vacuoles containing electron-dense material are present. Membrane is observed between adjacent vacuoles (arrow). No dilatation of the endoplasmic reticulum is observed. (Bar, 1 rm.) (D) Eighteen hours of incubation with H pylori supernatant results in cytoplasmic vacuoles of varying size containing abundant electron-dense material. Membranes lining the vacuoles are not evident. The contents of two of the vacuoles are confluent (arrow). (Bar, 1 pm.)

1006

H PYLORI CYTOTOXIN-INDUCED

FIGURE 1. (Continued) (E) After 6 days of incubation with Ii pylori supernatant numerous cytoplasmic vacuoles containing abundant electron-dense material are present, consistent with secondary lysosomes. Many vacuoles are surrounded by membrane. (Ear, 1 pm.)

VACUOLATION

(Cover et al)

FIGURE 3. Electron micrograph of an HeLa cell treated for 24 hours with MEM-FBS containing 25 mmol/L trimethylamine. Abundant vacuoles, some containing electron-dense debris, are present in the cytoplasm. Several of the vacuoles are membrane lined. (Bar, 1 +m.)

duced by Hpylori 60 190 supernatant or trimethylamine (Fig 4, top left and top right), but did not stain cells incubated in MEM-FBS alone (Fig 4, bottom). The vacuoles induced by H pylori supernatant were larger than those induced by trimethylamine, but the vacuoles were otherwise similar in appearance and staining characteristics. Neutral red is a weak base that crosses membranes in an uncharged state and concentrates in acidic cell compartments, such as lysosomes.20~22The rapid uptake of neutral red into H pylori supernatant-induced vacuoles indicated that the intravacuolar pH was acidic. Reversibility

FIGURE 2. Assessment of acid phosphatase activity in a vacuole induced by H pylori supernatant. HeLa cells were incubated for 6 hours in MEM-FBS containing a I:20 dilution of supernatant from H pylori strain 60190 and stained for acid phosphatase activity. as described in the Materials and Methods section. Markedly electron-dense reaction material is present within the vacuole. (Bar, 1 pm.)

of Vacuolation

To determine if the vacuolation induced by Hpylori supernatant was a reversible phenomenon, the medium overlying vacuolated cells was removed and the cells were washed and incubated with fresh MEM-FBS for 2 to 6 hours. Cell vacuolation was quantitated using a rapid neutral red uptake assay, which permitted assessment of the entire cell population.g The significant decreases in net optical density after media exchange indicate that the vacuolation induced by H pylori supernatant was a reversible phenomenon (Fig 5, left); vacuolation induced by trimethylamine was similarly reversible (Fig 5, right). Inspection of the cells by light microscopy confirmed these conclusions. Labeling of Cell Vacuoles With Lucifer Yellow To investigate further the relationship between H vacuoles and lysosomes, cel-

pylori supernatant-induced

HUMAN PATHOLOGY

Volume 23, No. 9 (September

1992)

FIGURE 4. Neutral red staining of HeLa cells. HeLa cells were incubated with MEM-FBS containing a I:20 dilution of concentrated supernatant from H pylori strain 60190 (top left) or MEMFBS containing 25 mmol/L trimethylamine (top right) for 18 hours at 37°C and then stained for 4 minutes with 0.05% neutral red dye. Neutral red avidly accumulated within the vacuoles induced by either H pylori supernatant or trimethylamine, but did not stain the cells incubated with MEM-FBS alone (bottom). (Magnifications: top left and top right, x107; bottom, ~198.)

lular endocytic compartments were labeled with lucifer yellow, a fluorescent marker for fluid-phase pinocytoCells were incubated in MEM-FBS containing sis. ’ b,23,24 Hpylori supernatant plus 100 pg/mL lucifer yellow, 25 mmol/L trimethylamine plus lucifer yellow, or lucifer yellow alone. When assessed by light microscopy, H&dori supernatant and trimethylamine each induced vacuolation of the entire cell population. However, a significantly greater proportion of trimethylamine-induced vacuoles were labeled by lucifer yellow than were Hpylori supernatant-induced vacuoles (Table 1); as expected, only rare fluorescent vacuoles were observed in control cells incubated with lucifer yellow alone. Thus, at an early stage in formation, vacuoles induced by trimethylamine and H pylori supernatant differed markedly in endocytic activity. Incubation with Hpylori supernatant for 18 hours was associated with an increased number of fluorescent vacuoles compared with 6-hour incubation (P < .OOOl) (Table l), indicating that with time the vacuoles became increasingly more accessible to the endocytic tracer. DISCUSSION The pathogenic mechanisms whereby H pylori infection gives rise to human disease remain poorly un1008

derstood. H pylori rarely invades the gastroduodenal mucosa and, hence, secreted products, such as urease, mucinase, phospholipase, and cytotoxins, may play a role in inciting epithelial cell damage. In addition, motility and adherence of the organism may be important in the initiation and maintenance of infection. Finally, recruitment and activation of leukocytes by H pylori surface proteins, including urease, is thought to play an important role in the pathogenesis of gastroduodenal inflammation. In this study the ultrastructure of Hpylori cytotoxininduced vacuoles was studied at a sequence of timepoints. At the earliest stages of formation, vacuoles were not clearly associated with any organelle. The intact structure of mitochondria, endoplasmic reticulum, and secondary lysosomes suggested that vacuoles did not arise directly from these structures. Lining membranes were not consistently observed in the early stages of H pylori supernatant-induced vacuoles; this phenomenon may be attributable to membranous fragility26*27 or may be an artifact of the fixation and cutting process. After prolonged incubation, vacuoles contained increasing quantities of debris. This suggested the occurrence of degradative processes and was consistent with the identification of acid phosphatase activity, a lysosomal enzyme, ” within the vacuoles.

H PYLORI CVTOTOXIN-INDUCED

A-A 0-O n -m O-O 0-O

0 2 6 2 6

h h h h h

VACUOLATION

A--A0 O-02

control control control removal removal

h h h h h

m-m6 O-02 O--O6

0 .u z

5o -0.1

(Cover et al)

control control control removal removal

0.1 --

O----_-o---O--------6 0 p-----6

0.0

L

I 0

-0.1

J 20

40

80 Reciprocal

160

25

320

12.5

6.25 Concentration

dilution

3.12

1.56

(mM)

FIGURE 5. Reversibility of vacuolation induced by H pylori supernatant (left) or trimethylamine (right). HeLa cells were incubated for 18 hours with MEM-FBS containing dilutions of concentrated supernatant from H pylori 60190 or 1.56 to 25 mmol/L trimethylamine, and then washed and incubated in MEM-FBS without additives for 2 to 6 hours. As a control cells were incubated for 2 to 6 additional hours without media exchange. Vacuolation was quantitated spectrophotometrically based on the uptake of neutral red into vacuoles. Vacuolation induced by either H pylori supernatant or trimethylamine was significantly diminished following incubation in fresh medium (P < .05)

We then compared the cell vacuolation induced by cytotoxin and trimethylamine. Trimethylamine is a weak base that crosses membranes in an uncharged state and is protonated and concentrated within acidic compartments of the cell, thereby inducing lysosomal swelling. 20,22 Neutral red dye, a lysosomotropic weak base, avidly accumulated in the vacuoles induced by either Hpylori cytotoxin or trimethylamine, an indication that the intravacuolar pH was acidic.‘* The vacuolation produced by both agents was reversible on incubation of the cells in fresh medium. At a morphologic level vacuoles induced by trimethylamine were smaller and more frequently contained intact lining membranes than vacuoles induced by H pylori supernatant. In addition, Hpylori cytotoxin- and trimethylamine-induced vacuoles differed significantly in uptake of lucifer yellow, a flu-

Hpylori

TABLE 1.

Time (W

6

18

Lucifer Yellow Uptake by Cell Vacuoles

No. of Fluorescent Vacuoles Per Cell 0 1-2 >2 0 l-2 >2

Percentage of Cells (Mean f SEM) Containing Specified No. of Fluorescent Vacuoles in Response to the Agent Tested* H f~ylori

Trimethylamine 3 3 94 2 2 96

+ + f f + +

1.5 0.8 2.3 0.6 0.9 1.5

Supernatant

MEM-FBS Alone

99 + 0.6 1 + 0.6 Ok0 80 f 2.4 16 -c 2.3 4 k 0.6

100 f 0.3 0 + 0.3 Ok0 99 + 0.6 1 f 0.6 020

* HeLa cells were incubated for 6 or 18 hours with MEM-FBS containing concentrated supernatant from H pylori 60190 plus lOOpg/mL lucifer yellow, 25 mol/L trimethylamine plus lucifer yellow, or lucifer yellow alone. The percentage of cells containing fluorescent vacuoles was then determined by visual inspection using fluorescent microscopy. At 6 hours, fluorescent vacuoles induced by trimethylamine were labeled by lucifer yellow to a significantly greater extent than vacuoles induced by H pylori supernatant (P < .OOOl, Fisher’s exact test).

1009

orescent endocytic tracer. Lucifer yellow is a soluble, anionic, low molecular weight, membrane-impermeable, fluorescent molecule that enters the endocytic compartment of cells by fluid-phase pinocytosis.‘6*23,24 In the early stages of formation, the majority of trimethylamine-induced vacuoles were labeled by lucifer yellow, whereas Hpylori supernatant-induced vacuoles were not. These data suggest that trimethylamine-induced vacuoles arise directly from endocytic compartments, whereas Hpylori cytotoxin-induced vacuoles do not. The identification of structures consistent with normal-sized secondary lysosomes in cytotoxin-treated cells containing nascent vacuoles provides further evidence that the vacuoles do not arise directly from lysosomes. The endocytic hypoactivity of early stage H pylori cytotoxin-induced vacuoles suggests that these vacuoles may arise via an autophagic mechanism. The earliest stages of autophagic vacuoles (autophagosomes) are typically endocytically inactive, but may become accessible to endocytic tracers following fusion with endosomes or lysosomes. 28.2gThe increased uptake of lucifer yellow by cytotoxin-induced vacuoles incubated for 18 hours compared with 6 hours may be explained by convergence of the autophagic and endocytic pathways.28 The determination that H pylori cytotoxin induces the formation of autophagic vacuoles provides insight into potential mechanisms whereby this toxin exerts its effect. The formation of autophagic vacuoles by agents such as leupeptin is associated with the specific inhibition of lysosomal proteases.‘s~‘g Autophagic vacuole formation by vinblastine is associated with impairment of microtubular function.1s,1g Future studies will determine whether H pylori cytotoxin induces cell vacuolation by analogous mechanisms. NOTE ADDED

IN PROOF

The vacuolating cytotoxin of H pylori recently has been purified and characterized.30

HUMAN PATHOLOGY Acknowledgment.

technical

assistance

The authors thank in electron microscopic

Stella Askins studies.

Volume 23. No. 9 (September

for

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15. bmashiro DJ. Maxfield FR: Acidification of morphologic alI\ distinct endosomes in mutant and wild-type Chinese hamster ovarb cells. J Cell Biol 105:2723-2733. 1987 16. DeCourcy K, Storrie B: Osmotic swelling of endocytic conpartments induced by internalized sucrose is restricted to mature lysosomes in cultured mammalian cells. Exp Cell Res 192:52-60, 199 1 17. Holtzman E: .4cid phosphatase as a “marker” enzyme for lysosomes, in HoltLman E (ed): Lysosomes. New York. NY, Plenum, 1989, pp 11-13 18. Glaumann H, Ahlberg J: Comparison of different autophagic vacuoles with regard to ultrastructure, enzymatic composition, and degradation capacity-formation of crinosomes. Exp Mel Pathol 47: 346-362, 1987 19. Kovacs AL, Laszlo l., Kovacs J: Effect of amino acids and cycloheximide on changes caused by vinblastine. leupeptin and methylamine in the autophagic/lysosomal system of mouse hepatocytes in vivo. Exp Cell Res 157:83-94, 1985 20. Ohkuma S, Poole B: Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances. J Cell Biol 90:656-664, 1981 2 1. Bulychev A, Trouet A, Tulkens P: Uptake and intracellular distribution of neutral red in cultured fibroblasts. Exp Cell Res 115: 343-355, 1978 22. Holtzman E: Acidification, membrane properties, permeability and transport, in Holtzman E (ed): Lysosomes. New York, NY, Plenum, 1989, pp 93-157 23. Swanson J: Fluorescent labeling of endocytic compartments. Methods Cell Biol 29:137-151, 1989 24. Swanson J. Yirinec B. Burke E, et al: Effect of alterations in the size of the vacuolar compartment on pinocytosis in 5774.2 matrophages. J Cell Physiol 128: 195-201, 1986 25. Mai UEH, Perez-Perez GI, Wahl LM, et al: Soluble surface proteins from Helicobacttr pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism. J Clin Invest 87:894-900, 1991 26. Deter RI., de Duve C: Influence of glucagon, an inducer of cellular autophagy, on some physical properties of rat liver lysosomes. J Cell Biol 33:437-449, 1967 27. Solheim AE, Seglen PO: Structural and physical changes in lysosomes from isolated rat hepatocytes treated with methylamine. Biochim Biophys Acta 763:284-291, 1983 28. Gordon PB, Seglen PO: Prelysosomal convergence of autophagic and endocytic pathways. Biochem Biophys Res Commun 15 1: 40-47, 1988 29. Tooze J, Hollinshead M, Ludwig T, et al: In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome. J Cell Biol 111:329345, 1990 30. Cover TL, Blaser MJ: Purification and characterization of the vacuolating toxin from Helico&acterf$ori. J Biol Chem 267: 1057010575, 1992

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