THE EFFECT OF Mn(II) ON THE AUTOINDUCING GROWTH INHIBITION FACTOR IN Deinococcus radiodurans

Decreases in cell division at the stationary phase in bacterial cultures are often due to the depletion of nutrients and/or accumulation of toxic waste products. Yet, during the stationary phase, the highly radiation-resistant bacterium Deinococcus radiodurans undergoes new rounds of cell division when Mn(II) is added to the medium in a phenomenon known as manganese-induced cell division (MnCD). When cells were cultured in medium without Mn(II)-enrichment, a heat-resistant, proteinase K-resistant factor (or factors) with a molecular mass less than 10 kD accumulated in the spent medium. Inclusion of the concentrated spent medium in fresh medium could inhibit the growth of D. radiodurans significantly, and the degree of inhibition was dose dependent. However, the relative stimulatory effect of MnCD was also dose dependent—the higher the inhibition, the stronger was the MnCD response. Previous studies have shown that nutrients were not limiting and deinococcal cells would continue metabolizing its nutrients at stationary phase. Cells became more sensitive to radiation when nutrients in the medium eventually became depleted. We speculated that D. radiodurans might produce this factor in the medium to control its population density. The reduction in cell population would conserve the nutrients that in turn might enhance the survival of the species.     

Role of DNA synthesis in secretion of immunoglobulin from murine B cells stimulated by T cell derived lymphokines

We have investigated whether cell division is required for induction of Ig secretion from three types of B cells, which represent distinct activation states: normal splenic B cells, anti-Ig-treated B cells, and a monoclonal murine B cell tumor, BCL1. Polyclonal Ig secretion was stimulated in vitro by LPS or by lymphokines produced by EL-4 cells (EL-4 SN), which includes B cell growth factor II (BCGF II). LPS and EL-4 SN were mitogenic for all three cell populations and stimulated substantial IgM secretion from both B cells and anti-Ig blasts. Aphidicolin, a reversible inhibitor of DNA synthesis, abolished IgM secretion from B cells and anti-Ig blasts induced by either mitogen, indicating that Ig-secreting cells in these cultures are part of a cycling population. BCL1 tumor cells respond to BCGF II (but not to interleukin 2 or B cell stimulatory factor 1) with IgM secretion and cell division, allowing a direct assessment of the influence of BCGF II-stimulated cell division on secretion of IgM. Secretion by these cells during the first 24 hr of culture was not substantially affected by aphidicolin, but secretion at 48 or 72 hr was markedly inhibited. Culture of BCL1 cells for 48 hr with aphidicolin alone had no effect on cell viability or on subsequent responsiveness if the drug was removed, eliminating non-specific toxicity as an explanation of the drug's effect. Addition of aphidicolin during the last 24 hr of culture to either normal B cells or BCL1 cells was much less effective at inhibiting IgM secretion. These results indicate that the cells that secrete IgM in response to BCGF II also synthesize DNA when exposed to this factor. Thus, induction of high-rate Ig secretion from murine B cells by some stimuli, including BCGF II, may require at least one round of cell division.     

Characterization of spatial growth and distribution of chondrocyte cells embedded in collagen gels through a stereoscopic cell imaging system

The stereoscopic image analysis of fluorescence-labeled chondrocyte cells for cytoplasm and nucleus was performed for the quantitative determination of spatial cell distribution as well as cell aggregate size in the collagen-embedded culture. The three-dimensional histomorphometric data indicated that the cells in the gels formed aggregates by cell division, and the size of aggregates increased with elapsed culture time. In the culture seeded at 2.0 x 10(6) cells/cm(3), the cells showed a semilunar shape that is a typical chondrocytic morphology, and formed the dense cell aggregates producing collagen type II. From the quantitative analysis of aggregate size, in addition, it was found that the cell division caused the aggregate growth with an increase of cell number in respective aggregates at 7 days, and some of aggregates made coalescence at 14 days. In the gel surface region, further coalescence of aggregates accompanied with cell division produced larger cell clusters, creating cell layers on the gel surface at the end of culture (21 days). In the culture seeded at 2.0 x 10(5) cells/cm(3), the different manner of aggregation was observed. At 14 days, the loose clusters of spindle-shaped cells emerged in the deeper region of gels, suggesting that the cell migration and gathering occurred in the gels. This loose-clustered aggregates did not produce collagen type II. Our results suggest that the seeding density is a factor to cause different mechanisms of cell distribution accompanied with the formation of aggregates as well as collagen type II.     

Adaptation to cycloheximide of macromolecular synthesis in Tetrahymena

Cycloheximide (CHI) at 10 ng/ml partially inhibited protein synthesis in exponential cultures of Tetrahymena Sp. At 20 ng/ml or greater, inhibition was complete. When protein synthesis was inhibited to any extent, cell division ceased immediately. In all instances where measured, synthesis of RNA and DNA also ceased. After a period of delay, cellular functions reinitiated in the order: (i) protein synthesis, (ii) DNA synthesis and, (iii) RNA synthesis and cell division. The delay in cell division was divided into three phases of: I, zero; II, low; and, III, fully recovered rates of exponential protein synthesis. The length of the three phases increased with increasing concentration of CHI Prior growth of cells for one generation in the presence of 7.5 ng/ml CHI (facilitation) eliminated phase I and slightly decreased phases II and III following subsequent challenge with an inhibitory concentration of CHI. Facilitation for six generations further decreased phases II and III. Protein synthesis and cell division were not inhibited during facilitation In the culture, succinate dehydrogenase activity did not increase during the delay but increased normally at the onset of division. In contrast, NADPH-cytochrome c reductase activity continued to increase for an hour after inhibition of protein synthesis, was constant for a period and did not increase again until an hour after reinitiatoin of cell division and RNA synthesis Inhibition of division of all cells was immediate and reinitiation of synthesis and cell division was non-synchronous.     

Apoptosis at inflection point in liquid culture of budding yeasts

Budding yeasts are highly suitable for aging studies, because the number of bud scars (stage) proportionally correlates with age. Its maximum stages are known to reach at 20-30 stages on an isolated agar medium. However, their stage dynamics in a liquid culture is virtually unknown. We investigate the population dynamics by counting scars in each cell. Here one cell division produces one new cell and one bud scar. This simple rule leads to a conservation law: "The total number of bud scars is equal to the total number of cells." We find a large discrepancy: extremely fewer cells with over 5 scars than expected. Almost all cells with 6 or more scars disappear within a short period of time in the late log phase (corresponds to the inflection point). This discrepancy is confirmed directly by the microscopic observations of broken cells. This finding implies apoptosis in older cells (6 scars or more).     

Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents

Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.     

Étude de l'effet de la température sur la division des cellules séparées de feuilles deCalystegia sepium (L.) R.Br.

Cells mechanically isolated from homogenized leaves ofC. sepium dividein vitro. The rate of cell division is strongly influenced by temperature with the optimum between 31 and 34 °C. The rate of cell division increases proportionally with a rise in temperature up to 31 °C and is accompanied by a reduction in the length of the preparatory phase of the division. A supraoptimal temperature (40 °C) inhibits or stops the cell division which can be restored provided that the cells are transferred to 31 °C. If the preparatory phase to the commencement of cell division is accomplished at 17 °C the cells then exposed to 31 °C divide more rapidly than if they are exposed directly to 31 °C.     

Inhibition of plastid division by ampicillin in the pteridophyte Selaginella nipponica Fr. et Sav

We investigated the effect of the beta-lactam antibiotic, ampicillin, on plastid division in the pteridophyte Selaginella nipponica. Guard cells of plantlets treated with 1 mM ampicillin only often had one plastid, whereas guard cells of untreated plantlets had two to four plastids. We generated a S. nipponica cell culture system and used it to investigate the effects of ampicillin. Treatment with 1 mM ampicillin had no effect on cell division in culture. We classified cultured cells into four types based on the number of plastids they contained: one (Type I), two (Type II), three or four (Type III) and more than five (Type IV). After 3 d in culture, the percentage of each cell type (I-IV) was 29.5, 46.7, 20.9, and 1.9%, respectively. Subsequently, the percentage of Types III and IV increased gradually, reaching 61.9 and 11.4%, respectively, after 15 d in culture in the absence of ampicillin. When 1 mM ampicillin was added, there was a minimal increase in the number of Type III and IV cells, with high percentages of Type I and II cells (32.4 and 45.7%, respectively) after 15 d. These results suggest that ampicillin inhibits plastid division in S. nipponica.     

Effect of cytomegalovirus on cell cycle progression and formation of pathological mitoses in cultured human diploid fibroblasts

The effect of cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G2, or M); at the same time, at least part of infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained 3H-thymidine label above chromosomes. This fact suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of cytomegalovirus.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

The role of auxin-binding protein 1 in the expansion of tobacco leaf cells

Tobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor.     

Cu(II) and Zn(II) ions alter the dynamics and distribution of Mn(II) in cultured chick glial cells

Previous studies revealed that Mn(II) is accumulated in cultured glial cells to concentrations far above those present in whole brain or in culture medium. The data indicated that Mn(II) moves across the plasma membrane into the cytoplasm by facilitated diffusion or counter-ion transport with Ca(II), then into mitochondria by active transport. The fact that 1–10 M Mn(II) ions activate brain glutamine synthetase makes important the regulation of Mn(II) transport in the CNS. Since Cu(II) and Zn(II) caused significant changes in the accumulation of Mn(II) by glia, the mechanisms by which these ions alter the uptake and efflux of Mn(II) ions has been investigated systematically under chemically defined conditions. The kinetics of [⁵⁴MN]-Mn(II) uptake and efflux were determined and compared under four different sets of conditions: no adducts, Cu(II) or Zn(II) added externally, and with cells preloaded with Cu(II) or Zn(II) in the presence and absence of external added metal ions. Zn(II) ions inhibit the initial velocity of Mn(II) uptake, increase total Mn(II) accumulated, but do not alter the rate or extent Mn(II) efflux. Cu(II) ions increase both the initial velocity and the net Mn(II) accumulated by glia, with little effect on rate or extent of Mn(II) efflux. These results predict that increases in Cu(II) or Zn(II) levels may also increase the steady-state levels of Mn(II) in the cytoplasmic fraction of glial cells, which may in turn alter the activity of Mn(II)-sensitive enzymes in this cell compartment.     

Synthesis of photoreactivating enzyme in synchronous and asynchronous cultures of Escherichia coli

The technique of flash photolysis was used to study cellular variations in the number of photoreactivating enzyme (PRE) molecules during the cell division cycle of the UV-sensitive E. coli strain B_S?1. No variations in the number of PRE molecules per genome were observed throughout the cell division cycle when synchronized cells cultured in either glucose-minimal or succinate-minimal medium were used. This is interpreted to mean that PRE synthesis is continuous throughout the cell cycle for glucose-grown cells, but may stop at the time chromosome replication ceases prior to division, in succinate-grown cells. The effect of growth rate and stage of growth on cellular PRE content in asynchronous cultures was also determined. Variations in the number of PRE per genome were observed for both synchronous and asynchronous cells cultured in different media and occurred in a manner that suggested a dependence on growth rate. PRE per genome increased with generation time. Stationary phase cells from each culture medium (nutrient broth, glucose-minimal, succinate-minimal) had more PRE per genome than did respective log phase cells. It is suggested that PRE synthesis may be controlled by some aspect of chromosome replication.     

Changes in cAMP and fibronectin concentrations in adherent and suspension forms of a hybrid cell line

The coexistence of adherent (PCM3-M) and suspension (PCM3-S) cells in a hybrid cell line led to the investigation of changes in cellular adhesion. The clonal origin, stable karyotype and subculturing properties of this hybrid indicate that these cells represent different forms of an identical population. Time-lapse photography and mitotic studies signify that the PCM3-S cells were found in the mitotic phase of the cell cycle, while incorporation of [³H]thymidine into DNA by both PCM3-M and PCM3-S cells suggested that the adherent cells detach during the S phase. This was substantiated by inducing quiescence of the culture during the G2 phase, since a preferential increase in the suspension was subsequently observed. Thus the cell cycle studies are consistent with the proposal that floating cells are discernable in a culture of adherent cells because the point at which they round up for division occurs earlier in the cell cycle. Prostaglandin E₁ (2.8 μM) and 3-isobutyl-1-methylxanthine (0.1 mM) were shown to maximally stimulate cAMP production and incubating the hybrid in these agents dramatically decreased the percentage of floating cells, slowed the growth rate and increased the adhesion of the PCM3-M cells. However, determination of intracellular concentrations of cAMP indicated that the floating cells were not a manifestation of decreased cAMP concentrations. In addition, the cell surface glycoprotein, fibronectin, was found to be present on the PCM3-M cells but lost from the PCM3-S cells. Addition of extracted fibronectin to these floating cells resulted in a culture consisting exclusively of adherent PCM3-M cells, suggesting that an aberration in fibronectin-associated cell-substratum adhesion is responsible for the unusual growth of the hybrid.     

Modulation of chondrocyte migration and aggregation by insulin-like growth factor-1 in cultured cartilage

The effect of insulin-like growth factor-1 (IGF-1) on the behavior of rabbit chondrocytes in cultured collagen (CL) gels initially seeded with 2 × 10⁵ cells/ml was examined. On day 5, the frequency of migrating cells cultured in presence of 100 ng IGF-1/ml was 0.04, which was 54 % of the frequency in IGF-1-free culture. The presence of IGF-1 caused an increase in the frequency of dividing cells from 0.09 to 0.13. These results suggest that IGF-1 suppressed the migration of chondrocytes in the CL gels while stimulating cell division in the initial culture phase. The proteolytic migration of cells was thought to be suppressed by the down-regulation of membrane type 1 matrix metalloproteinase by IGF-1. This contributed to the formation of aggregates with spherical-shaped cells that produced collagen type II.     

Regulation of gluconeogenic enzymes during the cell cycle of Saccharomyces cerevisiae growing in a chemostat

Oscillation of the activities of gluconeogenic enzymes (malate dehydrogenase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) was observed during the cell cycle of chemostat cultures of Saccharomyces cerevisiae. Since ethanol is released by the cells at the beginning of the division cycle, its effect on enzyme expression was determined. Pulsing ethanol to a synchronously dividing yeast culture led to a prolongation of the metabolically active phase as indicated by the course of oxygen uptake and carbon dioxide production rates (concomitant ethanol and glucose assimilation). Enzyme activities also remained elevated as long as ethanol was available to the cells. After a substrate shift from glucose to ethanol during cell division, ethanol was used without a lag phase and enzyme induction increased from the level reached at the point of the substrate change. The data confirmed that the small amount of ethanol produced when the cells begin active reproduction acts as an inducer of gluconeogenic enzymes.     

Effect of the Cytomegalovirus on Cell Cycle Progression and Pathological Mitoses in Cultured Human Diploid Fibroblasts

The effect of the cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G ₂, or M); at the same time, at least part of the infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained ³H-thymidine label above chromosomes. This suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of the cytomegalovirus.     

Influences of construct properties on the proliferation and matrix synthesis of dedifferentiated chondrocytes cultured in alginate gel

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.     

Characterization of transglutaminase activity in rabbit tracheal epithelial cells. Regulation by retinoids

Rabbit tracheal epithelial cells undergo terminal cell division, start to express a squamous phenotype, and form cross-linked envelopes when reaching the plateau phase of the growth curve. This terminal differentiation is accompanied by a 20-30-fold increase in the activity of the cross-linking enzyme transglutaminase. This activity is found almost solely in the particulate fraction of homogenized cells and can be solubilized by nonionic detergents. This transglutaminase crossreacts with a monoclonal antibody raised against type I transglutaminase, but does not react with an antiserum against type II transglutaminase. The tracheal transglutaminase contains a protein subunit of approximately 92 kDa. The omission of epidermal growth factor from the medium or the addition of fetal bovine serum, conditions that induce terminal cell division and expression of a squamous phenotype, enhance transglutaminase activity. High calcium concentrations only stimulate transglutaminase activity after the cells become committed to terminal cell division. Retinoids, which inhibit the expression of the squamous phenotype but not terminal cell division, inhibit the enhancement in transglutaminase activity induced by either confluency or serum, indicating that this enzyme activity is under the control of retinoids. Some retinoids are active at concentrations as low as 10(-12) M. The ability of retinoids to inhibit transglutaminase activity correlates well with their capacity to bind to the retinoic acid-binding protein. Our results show that the increase in transglutaminase activity correlates with the induction of the terminal differentiated phenotype and suggest that this enzyme can function as a marker for this program of differentiation of rabbit tracheal epithelial cells in culture. Our results identify the transglutaminase as type I transglutaminase and are in agreement with the concept that this transglutaminase is involved in the formation of cross-linked envelopes.