Effects of hyaluronidase, trypsin, and EDTA on surface composition and topography during detachment of cells in culture

Cultured human embryo fibroblasts (HLM18) were labeled with [3H]glucosamine and Na35SO4, and then treated with testicular hyaluronidase, trypsin, or EDTA. Macromolecular material from the surface of these cells was characterized by DEAE-cellulose chromatography and cetylpyridinium chloride precipitation while the associated morphology of cell detachment was studied by phase contrast and scanning electron microscopy. Release of surface glycosaminoglycans by testicular hyaluronidase did not cause cell rounding or detachment. EDTA did not release cell-surface components, but caused cell contraction and detachment morphologically similar to that caused by trypsin. Large amounts of cell-surface glycoproteins and glycosaminoglycans were released by trypsin. From these observations it is concluded that hyaluronic acid is not a principal adhesive agent in the attachment of cells to a substrate. It is suggested that both EDTA and trypsin may have their primary effect upon the cytoskeleton.     

Lysis of Vibrio succinogenes by ethylenediamine-tetraacetic acid or lysozyme

Wolin, M. J. (University of Illinois, Urbana). Lysis of Vibrio succinogenes by ethylenediaminetetraacetic acid or lysozyme. J. Bacteriol. 91:1781-1786. 1966.-Cell suspensions of Vibrio succinogenes are lysed by ethylenediaminetetraacetic acid (EDTA) or lysozyme. Lysis occurs at alkaline pH and is prevented by 0.15 m NaCl or KCl or 0.3 m sucrose. The addition of 10(-3)m Mg(++), 10(-3)m spermine, or 10(-2)m Ca(++) prevents lysozyme lysis, and 10(-4)m spermine prevents EDTA lysis. EDTA lysis leads to the formation of a cell ghost, and lysozyme lysis leads to the formation of an empty round body. Freezing and thawing of cells permits lysozyme attack which is not prevented by the protective agents mentioned above. Much of the cell protein, and almost all of the nucleic acids, are released from the cells during EDTA lysis. Treatment of frozen-thawed cells with lysozyme at neutral pH does not cause release of more than 50% of the cell protein and 60% of the nucleic acids of the cells.     

Comparative effects of trypsin, collagenase and mechanical harvesting on cell membrane lipids studied in monolayer-cultured endothelial cells and a green monkey kidney cell line

Experiments are presented in which membrane lipids of endothelial cells in monolayer culture were labelled with [14C]linoleic acid. Approx. 90% of the radioactive label were incorporated into phospholipids. A comparison of various harvesting methods showed that during the disruption of the labelled endothelial cell monolayer, 0.25% trypsin and 0.125% trypsin (+0.01% EDTA) released 650 and 470% more radioactivity, respectively, than did 0.01% collagenase (+0.01% EDTA). Parallel studies were performed on a green monkey kidney cell line. In this case, 0.25% trypsin released 520% more radioactivity than did 0.1% collagenase (+0.01% EDTA), although 0.125% trypsin in the presence of EDTA (0.01%) was much less traumatic than trypsin alone, the released radioactivity being of the same order of magnitude as that for collagenase. Morphological studies on endothelial cell cultures failed to reveal any distinctive differences in surface morphology following the various enzyme treatments. The results suggest that collagenase treatment of endothelial cell monolayers is the least traumatic harvesting or subculturing method as far as the integrity of the lipids in the cell membrane is concerned.     

Effects of Mitomycin C on Macromolecular Synthesis in Escherichia coli

When cells of Escherichia coli B growing in a glucose-synthetic medium were treated with mitomycin C, the effects produced by the antibiotic varied, depending on the concentration. When the concentration was reduced to less than 0.1 mug/ml, the action of the antibiotic was bacteriostatic; cell elongation resulted, but no effect on the synthesis of cellular macromolecules was apparent. At higher levels (more than 5 mug/ml), mitomycin C was highly bactericidal and inhibited deoxyribonucleic acid synthesis almost completely. The exposure of growing cells to a bactericidal level of mitomycin C resulted also in a delayed inhibition of the synthesis of ribonucleic acid (RNA) and protein. The capacity of the treated cells to synthesize beta-galactosidase inducibly in a medium free from a carbon source remained constant for the first 30 min and then was destroyed progressively with time. Prolonged incubation with the bactericidal level of mitomycin C caused a degradation of cellular nucleic acids, particularly RNA. The degraded nucleic acid components were eventually released into the medium.     

Factors affecting the binding of polycyclic aromatic hydrocarbons to human embryo cells, and transformable and non-transformable hamster embryo cells.

The effects of various factors, including population doubling number, percent of confluence, serum concentration and storage in liquid nitrogen on the binding of several polycyclic aromatic hydrocarbons to human and hamster embryo cells were studied. The binding of 7,12-dimethylbenz[a]-anthracene (DMBA) to hamster embryo cells DNA, RNA and protein was maximal after 22 h of treatment. In contrast, binding to human embryo cell macromolecules increased for at least 55 h. Treatment of hamster embryo cells at 100% confluence resulted in much less binding than treatment at 70% confluence, whereas with human embryo cells the binding increased, or remained constant, following treatment at the greater confluence. The transforming frequency of hamster embryo cells decreases with increasing population doubling number. Accordingly, we found that the binding of DMBA to hamster embryo DNA, RNA and protein decreased approximately 100-fold between population doubling numbers 8 and 20. In transformable cell cultures, DMBA was bound to hamster embryo cell DNA to a greater extent than to RNA or protein. The binding of DMBA to nucleic acids was much greater than binding by either dibenz[a,h]anthracene (DB[a,h]A) or dibenz-[a,c]anthracene (DB[a,c]A), both of which had low binding values at all population doubling numbers tested. Therefore, the best correlation of binding with carcinogenicity and transforming activity was observed with DMBA. Storage of hamster embryo cells in liquid nitrogen did not alter their binding characteristics. Binding of all three hydrocarbons to human embryo cell nucleic acids was low during all population doubling numbers studied, while binding to cellular protein increased until population doubling number 70 and then decreased sharply.     

Cell size and mutual cell adhesion. I. Increase in mutual adhesivenes of HeLa cells from density-inhibited suspension cultures by hypotonic treatment.

HeLa cells harvested from density-inhibited or fast growing suspension cultures, were incubated in NaCl solutions of different tonicity. Cell size enlargement produced by hypotonicity is accompanied by an increased sedimentation rate of the density-inhibited cells, whereas no appreciable change is observed in the sedimentation rate of fast growing cells. Hypotonicity also has no effect on the sedimentation rate of density-inhibited cells which previously had been treated with neuraminidase or trypsin. It is shown that the effect of hypotonicity on density-inhibited cells cannot be ascribed to release of cell surface sialic acids during hypotonic incubation. Several arguments are presented which indicate that the changes in sedimentation rate, as measured in the rotating suspension system, are not the direct consequence of the alterations in cell size, but rather must be attributed to differences in intercellular adhesiveness resulting from the size alterations. Analogous changes in intercellular adhesiveness and cell size are shown to occur during growth in isotonic suspension culture. The results can be explained by assuming that changes in cell size affect the intercellular adhesiveness by modifying the extent to which cell surface sialic acids counteract adhesion.     

Isolation and characterization of human cervical-mucus glycoproteins.

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 X 10(6) and 5.9 X 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).     

Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms

The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degrees C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physiochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms.     

Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms.

The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degrees C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physiochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms.     

Antibody dependent cell-mediated cytotoxicity of Trypanosoma cruzi: the release of tritium-labelled RNA, DNA and protein.

The cytotoxicity of normal rat spleen cells to antibody-coated Trypanosoma cruzi epimastigotes has been studied by assaying the release of [3H]-labelled macromolecules from the parasites. The release of thymidine (DNA) is slower than the release of uridine (RNA), suggesting that the nucleus is broken down more slowly than the cytoplasmic membrane. Less than 50% of the leucine (protein) is released when the parasites are lysed, whereas uridine (RNA) is almost totally released. In practical terms these results show that the release of incorporated radioisotope-labelled uridine can be used as a sensitive assay for cytotoxicity of T. cruzi. Cytotoxicity by normal rat spleen cells is antibody dependent and proportional to the logarithm of effector cell number. The lag phase and the rate of RNA release is not altered by centrifuging the parasites and effector cells to enhance contacts between them.     

Metabolic activities in human skin fibroblasts preloaded with labeled GM2-ganglioside

Confluent cultures of human skin fibroblasts were maintained for 10 days with sphingosine labeled [3H]GM2. Labeled medium was then replaced with normal medium and the cells maintained for 42 days with weekly medium changes. Cells were harvested at regular intervals and cells, medium, and trypsin digest supernatant analyzed for [3H]GM2 and its metabolic products. The ganglioside can be membrane associated and removed by trypsin, or membrane incorporated and trypsin insensitive. The membrane incorporated material is apparently transported to the lysosomes slowly by membrane flow, where 80% of the cellular GM2 can be metabolized by day 42. [3H]GM2 as well as its metabolic products in control cells is continuously released into the medium, during which it can also become associated with the cell surface membrane. There is no detectable metabolism of the [3H]GM2 in GM2 gangliosidosis cell lines over the extended post-labeling period, indicating that there is no residual enzyme activity in these cells. Undegraded GM2 is continuously released into the medium and remains associated with the cell surface membrane as well.     

Macromolecules released into the culture medium during the vegetative cell cycle of the unicellular green alga Chlamydomonas reinhardii.

The culture medium of growing Chlamydomonas reinhardii cells contains hydroxyproline-rich glycoproteins, which are mainly liberated during release of the zoospores from the mother-cell wall. Pulse-labelling studies with [3H]proline and [35S]methionine have been performed in order to detect the protein components released by synchronously growing cells at different stages of the cell cycle. When either [3H]proline or [35S]methionine were applied during the phase of cell growth, radioactive label appeared in the released macromolecules after a lag period of 40 min, whereas incorporation into the insoluble part of the cell wall was delayed only by 20 min. When applied at the end of the growth phase, e.g. 13 h after beginning of the illumination period, the radioactive amino acids were incorporated into the cell wall, but radioactive labelling of macromolecules released into the culture medium could not be detected before the zoospores were liberated from the mother-cell wall. Maximal incorporation of [3H]proline and [35S]methionine into the insoluble part of the cell wall was observed during cell division, but essentially no radioactively-labelled macromolecules were released into the culture medium during this time period. Analysis of the macromolecules, which were liberated during cell enlargement, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed distinct radioactive bands, which were differentially labelled with [3H]proline and [35S]methionine. Among the macromolecules released into the culture medium during cell growth, a component of an apparent Mr 35 000 was preferentially labelled with [3H]proline. This component was also detected after labelling with [35S]methionine, but components of an apparently higher Mr were more prominent after labelling with [35S]methionine. Macromolecules released during the cell-enlargement period of synchronously growing cultures in the presence of [3H]proline contained radioactively-labelled hydroxyproline in addition to proline. These results show that, during cell-wall growth, specific protein components are released into the culture medium and that at least one of these components contains large amounts of proline and hydroxyproline. At least some of these macromolecules seem to be constituents of the cell wall, because during pulse-chase experiments radioactively-labelled macromolecules appeared in the culture medium mainly during the time period when the specific radioactivity of the insoluble inner-cell-wall layer decreased.     

Organization of mammalian cytoplasm

Although the role of macromolecular interactions in cell function has attracted considerable attention, important questions about the organization of cells remain. To help clarify this situation, we used a simple protocol that measures macromolecule release after gentle permeabilization for the examination of the status of endogenous macromolecules. Treatment of Chinese hamster ovary cells with saponin under carefully controlled conditions allowed entry of molecules of at least 800 kDa; however, there were minimal effects on internal cellular architecture and protein synthesis remained at levels comparable to those seen with intact cells. Most importantly, total cellular protein and RNA were released from these cells extremely slowly. The release of actin-binding proteins and a variety of individual cytoplasmic proteins mirrored that of total protein, while marker proteins from subcellular compartments were not released. In contrast, glycolytic enzymes leaked rapidly, indicating that cells contain at least two distinct populations of cytoplasmic proteins. Addition of microfilament-disrupting agents led to rapid and extensive release of cytoplasmic macromolecules and a dramatic reduction in protein synthesis. These observations support the conclusion that mammalian cells behave as highly organized, macromolecular assemblies (dependent on the actin cytoskeleton) in which endogenous macromolecules normally are not free to diffuse over large distances.     

Glycine release from Y79 retinoblastoma cells

Abstract: Glycine release, induced by a high concentration of potassium chloride (K⁺), was investigated in cultured human Y79 retinoblastoma cells. The cells were labeled by incubation with [2-³H]glycine prior to K⁺ depolarization. Depolarization with 55 m M K⁺ caused an immediate, Ca²⁺-dependent release of approximately 20% of the cellular radiolabeled glycine content. Chemical analysis of the intracellular free glycine content also showed that approximately 20%, 2.4 nmol/mg protein, was released after K⁺ depolarization. Glycine release from labeled Y79 cells was not stimulated by incubation with 55 mM choline chloride. Based on measurements with an amino acid analyzer, it is concluded that of the free amino acids contained in the Y79 cell, only glycine is specifically released into the extracellular fluid by K⁺ depolarization. Although the intracellular content of serine and glutamate decreased, these amino acids were not released from the cells. Further studies with [U-¹⁴C]serine suggest that serine is converted into glycine in Y79 cells. Veratridine also caused an immediate release of [2-³H]glycine from the cells, and this was blocked by tetrodotoxin. This suggests that the Y79 cells possess voltage-dependent Na⁺ channels. These results indicate that K ⁺ - and veratridine-stimulated glycine release occurs in Y79 retinoblastoma cells, providing additional evidence that this continuously cultured line may be a useful model for certain human retinal and central nervous system functions.     

Studies on the Autolysis of Aspergillus oryzae

The conditions of autolysis of washed mycelia of Aspergillus oryzae were systematically examined as for temperature, pH, aeration, energy supply, and chemicals which stimulate autolysis. Below 45°C, the higher the temperature the faster was the rate of autolysis. Optimum pH of autolysis with special reference to the excretion of nucleic acid components and amino acids was 5. With the optimum conditions of autolysis settled by us, 90 to 100% of nucleic acids, 75% of protein, and 20% of sugars in the mycelia were excreted into the medium within three days.

In the presence of lipophilic compounds such as toluene and sodium salts of fatty acids, autolysis occurred much faster than in distilled water. Autolysis was inhibited by the addition of glucose and aeration.

Mycelia of Aspergillus oryzae were autolyzed in distilled water, in toluene-saturated water, or in acetate buffer, pH 5.4, at 30°C. The cytoplasmic materials disappeared from cells during autolysis, but the cell wall retained its shape even after autolysis. The disappearance of the cytoplasmic materials started from the inner part under an aerobic condition and from the outer part under an anaerobic condition. During the autolysis, 15% of the cellular proteins was excreted as free amino acids (60%) and peptides (15%). Glucose, ribose, glucosamine, and three unidentified sugars were found in autolyzate. After eighteen hours of autolysis stimulated by toluene, 81% of the cellular nucleic acids was excreted as uridine (28%), xanthine (24%), hypoxanthine (17%), and two other nucleosides or bases.     

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.     

In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus.

Low-temperature-induced nonculturable cells of the human pathogenic bacterium Vibrio vulnificus retained significant amounts of nucleic acids for more than 5 months. Upon permeabilization of fixed cells, however, an increasing number of cold-incubated cells released the nucleic acids. This indicates substantial degradation of DNA and RNA in nonculturable cells prior to fixation. Treatment of permeabilized cells with DNase and RNase allowed differential staining of DNA and RNA with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy revealed that the could-induced nonculturable populations of V. vulnificus are highly heterogeneous with regard to their nucleic acid content. The fraction of nonculturable cells which maintained DNA and RNA structures decreased gradually during cold incubation. After 5 months at 5 degrees C, less than 0.05% of the cells could be observed to retain DNA and RNA. In parallel with the loss of nucleic acids, an increase in the concentrations of UV-absorbing material in the culture supernatants was observed in nonculturable-cell suspensions. It is hypothesized that there are two phases of the formation of nonculturable cells of V. vulnificus: the first involves a loss of culturability with maintenance of cellular integrity and intact RNA and DNA (and thus possibly viability), and the second is typified by a gradual degradation of nucleic acids, the products of which partly remain inside the cells and partly diffuse into the extracellular space. A small number of nonculturable cells, however, retain DNA and RNA, and thus may be viable despite having reduced culturability.     

Disposition of glycoproteins in plasma membrane of cultured rat embryonic fibroblasts

Plasma membrane fractions were isolated from untreated and trypsin- or neuraminidase-treated rat embryo fibroblasts and their sialic acids contents per mg membrane protein were determined. The difference represented enzyme releasable sialic acid exposed on the medium side of the cell mambrane. It was 14 to 23% of the total membrane bound sialic acid. Isolated plasma membrane fraction from entreated and enzyme treated cells were then subjected to trypsin or neuraminidase treatment to obtain enzyme-releasable sialic acid from both faces and from the cytoplasmic face of the membrane respectively. Between 30 and 50% of the total membrane bound sialic was released from both the faces and 14 to 30% from the cytoplasmic face. An average of 59% was insusceptible to these enzymes. As an alternative to a cytoplasmic location of sialic acid containing membrane constituents, inaccessibility of enzymes to some of these constituents present on the surface of intact cells is considered.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of plasma membrane fractions isolated from untreated and trypsin treated cells and of trysinized plasma membrane fraction was carried out to know the number and gel migration of proteins and glycoproteins which are exposed on each of the two faces of the plasma membrane and are sensitive or insensitive to trypsin. The resilts obtained were confirmed by SDS-polyacrylamide gel electrophoresis of untreated and trypsin-treated cells and of isolated plasma membrane fraction after subjecting them to enzymatic radioiodination.     

Lysosomal degradation of glycoproteins and glycosaminoglycans. Efflux and recycling of sulphate and N-acetylhexosamines.

Lysosomal degradation of the carbohydrate portion of glycoproteins and glycosaminoglycans produces monosaccharides and sulphate, which must efflux from the lysosomes before re-entering biosynthetic pathways. We examined the degradation of glycoproteins and glycosaminoglycans by lysosomes isolated from cultured human diploid fibroblasts. Cells were grown for 24 h in medium containing [3H]glucosamine and [35S]sulphate. When lysosomes are isolated from these cells, they contain label primarily in macromolecules (glycoproteins and glycosaminoglycans). Glycoprotein degradation by isolated lysosomes was followed by measuring the release of tritiated sugars from macromolecules and efflux of these sugars from the organelles. Glycosaminoglycan degradation was monitored by the release of both tritiated sugars and [35S]sulphate. During macromolecule degradation, the total amounts of free [35S]sulphate, N-acetyl[3H]glucosamine and N-acetyl[3H]galactosamine found outside the lysosome parallels the amounts of these products released by degradation. The total degradation of glycoproteins and glycosaminoglycans by intact cultured cells was also examined. The lysosomal contribution to degradation was assessed by measuring inhibition by the lysosomotropic amine NH4Cl. After 48 h incubation, inhibition by NH4Cl exceeded 55% of glycoprotein and 72% of glycosaminoglycan degradation. Recycling of [3H]hexosamines and [35S]sulphate by intact cells was estimated by measuring the appearance of 'newly synthesized' radioactively labelled macromolecules in the medium. Sulphate does not appear to be appreciably recycled. N-Acetylglucosamine and N-acetylgalactosamine, on the other hand, are reutilized to a significant extent.