Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride

Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen.

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.     

The intracellular localisation of proteoglycans and their accumulation in chondrocytes treated with monensin

Pig laryngeal chondrocytes incubated in the presence of monensin showed inhibition of [³⁵S]sulphate incorporation and decreased secretion of proteoglycan into the culture medium, but no large decrease in protein synthesis. This lead to the intracellular accumulation of proteoglycan protein core, which was detected in immunoprecipitates of cell extracts. Using the same antiserum protein core was localised by electron microscopy with protein A-coated gold. In control chondrocytes, it was detected only in elements of the Golgi and in secretory vesicles, but following monensin treatment labelling was more intense in the Golgi and extended into the distended cisternae of the rough endoplasmic reticulum. The results suggest that monensin blocks proteoglycan protein core translocation between different elements of the Golgi and that this occurs prior to the major site of chondroitin sulphate synthesis on proteoglycan.     

Synthesis and secretion of the enamel matrix precursor by the kitten secretory ameloblast

Secretory ameloblasts in kitten molar tooth germs were examined with an electron microscope to analyze the synthesis and secretion processes of the enamel matrix precursor. The contents of the secretion granule were identified as fine granular material, which observed in both the rough endoplasmic reticulum and the Golgi cisterns, accumulated in the dilated margins of the innermost Golgi cistern and formed condensing vacuoles. The same kind of condensing vacuoles was also produced from the GERL cisterns. During the secretion granule maturation processes in the Golgi region, the contents accumulated densely and the granules grew smaller. In addition, granule-limiting membranes acquired fine, bristle coats. The mature secretion granules then migrated, along microtubules, into the surfaces of the Tomes processes and finally released their contents by a process of exocytosis at the type 1 face which faces the enamel growth region.     

Proteoglycan synthesis in suspension cultures of Swarm rat chondrosarcoma chondrocytes and inhibition by exogenous hyaluronate

Conditions were established for short-term primary suspension culture of chondrocytes from the Swarm rat chondrosarcoma. Proteoglycan and hyaluronate synthesis on Day 0 to Day 2 in culture was investigated and compared with that for plated cultures. Incorporation of [35S]sulfate into proteoglycans was the same for both suspension and plated cultures. 35S-Proteoglycan synthesis decreased by about 80% between Days 0 and 1 irrespective of culture conditions. Suspension culture chondrocytes synthesized proteoglycans which were very similar to those made in plated cultures, with respect to hydrodynamic size, glycosaminoglycan, chain length, and composition. [3H]Hyaluronate synthesis accounted for 18 and 23% of the total 3H-glycosaminoglycans synthesized from [3H]glucosamine by suspension and plated cultures, respectively. Suspension culture chondrocytes responded to exogenous hyaluronate (1 mg/ml) by reducing their 35S-proteoglycan synthesis by about 50%. [3H]Hyaluronate synthesis was inhibited by 13% under these conditions. The inhibition was dependent on the concentration of exogenous hyaluronate and reached a plateau level within 2 h. Plated chondrocyte cultures showed little or no response to hyaluronate. Suspension cultures of chondrocytes were prelabeled with [3H]lysine and lysed, and a heavy membrane fraction (12,000g) was extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. A Sepharose-hyaluronate affinity gel was used to show that the extract contained hyaluronate binding 3H-labeled proteins and evidence was obtained suggesting that these came from the external face of the plasma membrane.     

The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma

Proteoglycan synthesis by cultured chondrocytes from the Swarm rat chondrosarcoma was examined after treatment with 0.1 mg/ml of cycloheximide which inhibited [3H]serine incorporation into total protein by greater than 90%. Incorporation of [35S]sulfate into proteoglycans decreased with nearly first order kinetics (t 1/2 = 96 +/- 6 min) with an accompanying increase in the size of the proteoglycan molecules, primary due to an increase in chondroitin sulfate chain sizes. After 5 h of cycloheximide treatment, when [35S]sulfate incorporation was inhibited by about 90%, addition of 1 mM beta-D-xyloside restored 76% of the incorporation into chondroitin sulfate observed in cultures treated only with xyloside. This suggests that the biochemical pathways for the affected by cycloheximide treatment. Cultures were prelabeled for 15 min with either [3H]serine or [35S]-methionine, and then cycloheximide was added to block further protein synthesis. Both precursors appeared in completed proteoglycan molecules with nearly first order kinetics with t 1/2 values of 92 +/- 8 and 101 +/- 11 min for [3H]serine and [35S]methionine, respectively, values in close agreement with the t 1/2 from the [35S]sulfate data. These results suggest that after cycloheximide treatment, the rate of [35S]sulfate incorporation into proteoglycan, after a correction for increases in chondroitin sulfate chain size, was directly proportional to the size of the intracellular pool of core protein. From the steady state rate of proteoglycan synthesis (estimated to be about 80 ng/min/10(6) cells in separate experiments) and a corrected t 1/2 value of 60 min, the amount of precursor core protein can be calculated to be about 500 ng/10(6) cells in these experiments.     

Calcium and the secretory cycle of prolactin cells: a cytochemical and ultrastructural study of dopamine inhibition and monobutyryl cyclic AMP-stimulation of prolactin secretion

To identify intracellular calcium pools that may be involved in the secretory process in prolactin (PRL) cells, hemi pituitaries were incubated in medium containing 10(-6) M dopamine, 5 mM cyclic cAMP (experimentals), or in medium alone (controls) and then processed for electron microscopy using potassium pyroantimonate to localize intracellular calcium. PRL in the medium was measured by radioimmunoassay. The concentration of antimonate associated with mitochondria, Golgi saccules, and secretory granules was estimated. Dopamine inhibition of PRL secretion (> 80% at 1, 2, 3 h) resulted in accumulation of secretory granules in all stages of maturation and dilation of Golgi saccules at 2 and 3 h, accompanied by increased mitochondria antimonate and increased Golgi-associated antimonate. Cyclic AMP stimulation of secretion (635% at 5 min., declining to 34% at 1 h) resulted in marked exocytosis at 5 and 15 min., declining after 30 min. Mitochondrial antimonate decreased after 30 min. Stimulated cells exhibited numerous coated membrane structures at or near exocytotic pits and an amassing of microvesicles at the margin of the Golgi apparatus. Although some secretory granules consistently exhibited reactivity to antimonate (unchanged by inhibition or stimulation), plasma membrane, and granule membrane translocated to the plasma membrane during exocytosis, were not reactive.     

Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells

The synthesis and secretion of apolipoprotein B-100 (apoB-100) have been studied in a human hepatoma cell line, the Hep G2 cells. The time needed for the synthesis of apoB-100 was estimated to be 14 min, which corresponds to a translation rate of approximately 6 amino acids/s. ApoB-100 was compared with albumin and alpha 2-macroglobulin as to the distribution between the membrane and the luminal content in the endoplasmic reticulum (ER) and the Golgi apparatus. The results suggested that apoB-100 approximately followed the distribution of these secretory proteins in the Golgi, while the ratios between the percent membrane-bound apoB-100 and percent membrane-bound albumin or alpha 2-macroglobulin were 3-4:1 in the ER. This may suggest that apoB-100 occurs in a membrane-associated form in ER prior to the integration in the lipoproteins. Pulse-chase studies combined with subcellular fractionation was used to investigate the kinetics for the intracellular transfer of apoB-100. A 3-min pulse of [35S]methionine was followed by an increase in apoB-100 radioactivity in the ER during the first 10-15 min of chase. The following 10-15 min of chase were characterized by linear decrease in apoB-100 radioactivity with a decay rate of approximately 6%/min. The residence kinetics for apoB-100 in the ER differed from that of transferrin and probably also from that of albumin. By comparing the time for the pulse maximum in ER with that in the denser Golgi fractions the time needed for the transfer between ER and Golgi could be estimated to be 10 min. The time needed for the secretion of newly synthesized apoB-100 was estimated to be 30 min. This indicates that the transfer of the protein through the Golgi apparatus to the extracellular space requires 20 min.     

Proteoglycan metabolism associated with mouse metanephric development: morphologic and biochemical effects of beta-D-xyloside

Morphology and de novo incorporation of [35S]sulfate into proteoglycans were studied in fetal mouse kidneys at the onset of organogenesis. Branching morphogenesis and nephron development in organ culture and in vivo were associated with de novo synthesis of chondroitin-SO4 and heparan-SO4 proteoglycans. The role of proteoglycan metabolism in metanephrogenesis was then studied by analysis of the effects of p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside) on renal development and proteoglycan metabolism. Incubation of fetal kidneys in beta-D-xyloside at concentrations of 1.0 and 0.5 mM, but not at 0.1 mM, caused inhibition of ureteric branching and markedly diminished synthesis of a large Mr 2.0 X 10(6) Da chondroitin-SO4 proteoglycan. Incorporation of [35S]sulfate was stimulated at all beta-D-xyloside concentrations, reflecting synthesis of xyloside initiated dermatan-35SO4 chains. In contrast to dramatic effects on chondroitin-SO4 synthesis and ureteric branching, beta-D-xyloside had no effect on heparan-SO4 synthesis or on development of the glomerulus and glomerular basement membrane. We thus characterize the proteoglycans synthesized early in the course of renal organogenesis and describe observations which suggest an association between metabolism of chondroitin-SO4 proteoglycan and development of the ureter.     

Influence of colchicine on the synthesis and secretion of proteoglycans and collagen by fetal guinea pig chondrocytes

Fetal guinea-pig epiphyseal chondrocytes were cultured in monolayers and as aggregates in the presence of antimicrotubular agents. Colchicine and vinblastine caused a dissociation of the Golgi complex, in addition to the disappearance of microtubules. Synthesis and secretion of proteoglycans and collagen were studied using radioactive precursors. Colchicine inhibited the synthesis of proteoglycans. The drug also inhibited secretion with an intracellular accumulation of these molecules. The proteoglycans secreted by the colchicine-treated cells had a smaller molecular size and contained a smaller proportion of aggregated molecules than proteoglycans in control cultures. However, there was no difference in the average size of the chondroitin sulfate side chains of the proteoglycan molecules. Nor was there any increase in the breakdown of proteoglycans in colchicine-treated cultures. Vinblastine was also found to inhibit synthesis and secretion of proteoglycans. Deuterium oxide also inhibited the synthesis of these molecules but stimulated their secretion into the medium. Colchicine caused an inhibition of both synthesis and secretion of collagen. It is suggested that the quantitative and qualitative effects of colchicine could be the result of disturbances in the Golgi complex, possibly in combination with a retarded translocation of secretory vacuoles. However, as the colchicine-treated chondrocytes were still able to continue a large part of their matrix biosynthesis with only moderate changes in the structure of the secreted molecules, it is probable that alternative pathways for the secretion of matrix molecules exist and/or the Golgi complex is able to retain a major part of its function despite the structural alterations.     

SULFATE METABOLISM IN PANCREATIC ACINAR CELLS

The metabolism of inorganic sulfate in pancreatic acinar cells was studied by electron microscope radioautography in mice injected with sulfate-³⁵S. Labeled sulfate was concentrated in the Golgi complex at 10 min. Within 30 min, much of the radioactive material had been transferred to condensing vacuoles. These were subsequently transformed into zymogen granules. By 4 hr after injection, some of the zymogen granules with radioactive contents were undergoing secretion, and labeled material was present in the pancreatic duct system. The Golgi complex in pancreatic acinar cells is known to be responsible for concentrating and packaging digestive enzymes delivered to it from the endoplasmic reticulum. Our work demonstrates that the Golgi complex in these cells is also engaged in the manufacture of sulfated materials, probably sulfated mucopolysaccharides, which are packaged along with the enzymes in zymogen granules and released with them into the pancreatic secretion.     

Thyroglobulin is selected as luminal protein cargo for apical transport via detergent-resistant membranes in epithelial cells

Thyroid hormone synthesis by thyrocytes depends upon apical secretion of thyroglobulin (Tg), the glycoprotein prohormone. In stably transfected MDCK cells, recombinant Tg is also secreted apically. All secreted Tg has undergone Golgi carbohydrate modification, whereas most intracellular Tg (which is slow to exit the endoplasmic reticulum) is sensitive to digestion with endoglycosidase H. However, in MDCK cells and PC Cl3 thyrocytes, a subpopulation of newly synthesized recombinant and endogenous Tg, respectively, is recovered in a Triton X-100 insoluble, glycosphingolipid/cholesterol-enriched (GEM/raft) fraction, and this small subpopulation is overwhelmingly endoglycosidase H resistant. Upon apical secretion, Tg solubility is restored. Apical secretion of Tg is inhibited by cellular cholesterol depletion. In FRT cells, recombinant Tg becomes Triton X-100 insoluble within 60 min after synthesis and a portion is actually endoglycosidase H-sensitive, suggesting early Tg entry into GEMs/rafts. Interestingly in FRT cells, Tg remains associated with the apical plasma membrane upon exocytosis, and all surface Tg is GEM/raft-associated. Thus, Tg is the first secretory protein demonstrated to enter Triton X-100 insoluble membranes en route to the apical surface of epithelial cells. The data imply that Tg utilizes a cargo-selective mechanism for apical sorting.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHIC DETECTION OF SITES OF PROTEIN SYNTHESIS AND MIGRATION IN LIVER

The sites of synthesis of proteins and their subsequent migration in rat liver have been studied during a 75 min period after labeling of liver-slice proteins by exposure to leucine-H³ for 2 min. Incorporation of the label into protein began after 1 min and was maximal by 4 min. Electron microscopic radioautography showed that synthesis of proteins in hepatocytes occurs mainly on ribosomes, particularly those in rough endoplasmic reticulum and, to some extent, in nuclei and mitochondria. Most of the newly formed proteins leave the endoplasmic reticulum in the course of 40 min, and concurrently labeled proteins appear in Golgi bodies, smooth membranes, microbodies, and lysosomes. A likely pathway for the secretion of some or all plasma proteins is from typical rough endoplasmic reticulum to a zone of reticulum which is partially coated with ribosomes, to the Golgi apparatus, and thence to the cell periphery. The formation of protein by reticuloendothelial cells was measured and found to be about 5% of the total protein formed by the liver.     

Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae

Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.     

Vacuolated prolactin cells in the pituitary gland of several marine teleosts

A morphometric study of prolactin cell ultrastructure in the pituitary gland of the Corkwing wrasse, Crenilabrus melops L., showed that cytoplasmic vacuoles, which accounted for 25% of the cell volume, were associated with signs of decreased secretory activity. The Golgi apparatus, mitochondria and rough endoplasmic reticulum, all contributed relatively little to the total cell volume, and there was no sign of secretory-granule release by exocytosis. All vacuoles were intracellular and membrane-bound, and probably derived from rough endoplasmic reticulum, Golgi apparatus and the nuclear envelope. It is thought that smaller vacuoles coalesce to form larger ones. The secretory granules were small and sparse, and this could account for the chromophobia of prolactin cells in light-microscopy preparations. Similar vacuoles were reported in the prolactin cells of the gobiid fish, Chaparrudo flavescens, Pomatoschistus pictus, Pomatoschistus minutus and Pomatoschistus microps . The vacuoles in Chuparrudo were of similar ultrastructure to those in Crenilabrus .     

Light and electron microscopic observations of fabrication, release, and fate of biphasic secretion granules produced by epididymal epithelial principal cells of the fan-throated lizard Sitana ponticeriana cuvier

The epididymis of the fan-throated lizard Sitana ponticeriana was examined with light and transmission electron microscopy to understand the cellular mechanisms of fabrication of secretion granules in epithelial principal cells, granule release into the lumen, and the fate of the dense structured granules after reaching the lumen. Principal cells of the ductus epididymis, except at the cauda, secrete electron-dense biphasic granules copiously, which decrease in abundance from the initial segment to corpus. The principal cell possesses a prominent Golgi apparatus and all versions of endoplasmic reticulum (ER), rough, smooth, and sparsely granulated. The material of the dense portion of the secretion granules, after processing at the Golgi apparatus, appears to accumulate in large ER cisternae in the supranuclear cytoplasm. It undergoes condensation when the cisternae become condensing vacuoles. Mitochondria appear to play a role in dense granule formation. The condensing vacuoles are displaced toward the apical cytoplasm when the material of the less dense portion is added to the condensing vacuoles at the Golgi area. Thus, the less dense and dense portions of the secretion granules are secreted and added to the condensing vacuoles separately. The composite granules are released into the lumen by exocytosis when the less dense portion merges with the luminal content, whereas the dense portion maintains its structured identity. The latter, initially measuring 1-2 microm in diameter, increases in size several times. It is inferred that these granules release their content gradually, resulting in the appearance of vacuoles, and suggesting that the granules have an insoluble matrix in which there is a sparingly soluble material. The substance leaching out of the granules appears to contribute to keeping the sperm quiescent and alive during storage in the male reproductive tract.     

Ammonium chloride, methylamine and chloroquine reversibly inhibit antibody secretion by plasma cells

The effects of the lysosomotropic weak bases, NH4Cl, methylamine and chloroquine, on the secretory process of antibody-synthesizing cells were studied. Popliteal lymph node cells taken from rats immunized against horseradish peroxidase (HRP) were incubated with the lysosomotropic agents. The rate of secretion of anti-HRP antibodies was measured using an indirect enzyme-linked immunosorbent assay. These agents induced an inhibition of antibody release within 5 min, and for all four concentrations tested, maximal inhibition was reached after 15 min. A 50% inhibition was obtained with 20 mM NH4Cl, 21.7 mM methylamine and 8.8 X 10(-4) M chloroquine. This effect was rapidly and entirely reversible, regardless of the weak base used, and it increased as the pH of the extracellular media was raised. Under these conditions, intracellular ATP contents remained normal, and protein synthesis did not undergo marked changes except with chloroquine. Inhibition of secretion was accompanied by an intracellular accumulation of antibodies which was equal to the degree of inhibition of antibody release. Immunocytochemical studies of the weak base-treated cells performed by light and electron microscopy showed that this accumulation probably occurred within certain dilated Golgi saccules. In addition, reduced incorporation of fucose into immunoglobulins as well as partial inhibition of the secretion of fucosylated immunoglobulins were observed in the presence of weak bases. These results are consistent with the hypothesis that weak bases inhibit antibody secretion by acting within saccules of the Golgi apparatus. These saccules could maintain an acidic pH important for the migration and/or sorting of immunoglobulins.     

EFFECTS OF ANTIMICROTUBULAR AGENTS ON THE SECRETION OF COLLAGEN : A Biochemical and Morphological Study

Embryonic chick cranial bone was cultured in the presence of the antimicrotubular agents, colchicine and vinblastine, and with a number of other compounds known from previous studies to affect the cellular handling of collagen. Secretion of procollagen, quantitated by light microscope autoradiography, was correlated with the extent of conversion of procollagen to collagen and with rates of collagen and noncollagen-protein synthesis. Colchicine inhibited procollagen secretion and conversion to collagen and specifically inhibited collagen synthesis. Cells exposed to colchicine revealed an increased number of dilated Golgi-associated vacuoles and vesicles, some of which contained parallel aggregates of filamentous structures. These observations suggest that the pathway of at least a fraction of procollagen secretion by osteoblasts includes the Golgi complex. Disruption of microtubules may interfere with the movement of Golgi-derived vesicles, and the resulting accumulation of collagen precursors in the Golgi complex may lead secondarily to an inhibition of synthesis. Although vinblastine also inhibited both procollagen secretion and conversion to collagen, the observed reduction in general protein synthesis and striking changes in the ultrastructure of the rough endoplasmic reticulum complicated interpretation of the effects. Interpretation of the effects of cytochalasin B was limited by the finding that the cellular response in cranial bone was markedly heterogeneous and that, contrary to some previous reports, the drug caused an inhibition in the incorporation of radiolabeled amino acids into both collagen and noncollagen protein.     

The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes

Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 micron diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.     

Lysozyme expression in the rat parotid gland: light and electron microscopic immunogold studies

 Lysozyme (muramidase) is capable of direct bacteriolytic action by hydrolyzing glycosidic bonds in bacterial cell walls. Although it is broadly distributed in vertebrate tissues and secretions, the cellular and subcellular localizations of the enzyme are still not well known. The present study examines the distribution of lysozyme expression in the various cell types of LR gold-embedded rat parotid gland, applying a postembedding immunogold-silver staining technique for light microscopy. Simultaneously, a postembedding immunogold method for electron microscopy was used to determine the cellular compartments engaged in the biosynthesis and exocytosis of lysozyme. Silver-amplified immunogold staining for lysozyme demonstrated identical localization in both paraffin and semithin LR-gold sections: in the supranuclear parts of acinar and intercalated duct cells. Staining intensity varied even between adjacent cells. In the electron microscope, immunogold labeling was detected over the cell compartments associated with protein synthesis and exocytosis in acinar and intercalated duct cells. Lysozyme antigenic sites were visible over endoplasmic reticulum and throughout the Golgi apparatus, being intense over the trans-Golgi network, but even stronger in the condensing vacuoles and most prominent over secretory granules in both cell types. The findings provide the first immunocytochemical evidence of the synthesis and secretion of lysozyme in parotid acinar and intercalated duct cells. Accepted: 3 December 1996