Involvement of smooth muscle cells in collagen degradation in the postpartum uterus

Ultrastructural study of gravid and postpartum involuting human uteri revealed a number of cells containing collagen fibrils in their cytoplasm. In gravid uteri these cells could be identified as macrophages and fibroblasts; in the postpartum uteri smooth muscle cells (SMC) were also found, containing cytoplasmic collagenous vacuoles. The morphology of intracellular collagen in SMC was similar to that observed in macrophages: fragments of banded collagen fibrils with a diameter corresponding to that of extracellular collagen were located within structures considered to be phagosomes. Limiting membranes were always smooth, most often in apposition to the fibrils that were single or packed in small groups; some cytoplasmic vacuoles contained banded elongated profiles barely discernable as collagen. The collagen fibrils within SMC of the involuting human uterus are regarded as a morphological manifestation of heterogenic enclosure of collagen fibrils and their intracellular degradation. It seems that in the postpartum uterus, where a substantial amount of collagen needs to be removed rapidly, both macrophages and SMC are involved in the process of collagen phagocytosis and degradation. These data suggest that SMC may be involved in the cellular mechanism for collagen breakdown in remodelling SMC-containing tissues like the uterus and the vascular wall.     

Fine structural changes in uterine smooth muscle and fibroblasts in response to estrogen

The fine structure of the estrogen-primed uterus was examined in two series of rats, with emphasis upon the alterations in smooth muscle cells and fibroblasts. The first series of animals were mature animals that were sacrificed at diestrus or estrus. The second series consisted of prepubertal rats (57–70 g) that received subcutaneous injections of estradiol-17 β in 20% alcohol. Four groups of animals received the hormone twice daily for 3 days for a total dose of 0.06, 0.6, 6.0, or 60.0 µg, respectively. An estrogenic response was observed in all groups as indicated by an increase in uterine weight. Control groups consisted of either untreated animals or animals receiving 20% alcohol. All animals were sacrificed on the 4th day. The fibroblasts and smooth muscle cells in the controls were similar to their counterparts in the mature animal in diestrus. They were small, contained relatively little rough endoplasmic reticulum, and the connective tissue cells appeared like fibrocytes. All of the estrogen-treated animals were similar in appearance and were comparable to their counterparts in the mature animal in estrus. Both the smooth muscle cells and the fibroblasts were increased in size, demonstrated a marked enlargement and dilation of ergastoplasmic cisternae, and contained increased numbers of attached and free cytoplasmic ribosomes. The presence of an extensive rough endoplasmic reticulum in the smooth muscle cells of the stimulated uterus is in marked contrast to the appearance of these cells in other tissues. These observations correlate with previous biochemical studies by other workers, in which estrogens have been shown to promote the synthesis of uterine RNA, collagen, and noncollagenous protein, and suggest that smooth muscle cells may participate in the synthesis of connective tissue proteins.     

Collagenase production by smooth muscle: correlation of immunoreactive with functional enzyme in the myometrium

A monospecific antibody to rat uterine collagenase has been produced and employed to study the cell of origin and the time course of production of this enzyme in the involuting rat uterus. The specificity of the anti-collagenase antibody was confirmed by immunoprecipitation, Western analysis, and by its ability to inhibit the activity of collagenase. Parallel measurements of functional enzyme, both latent and active, bound to tissue collagen were also made in nonpregnant, gravid, and postpartum rat uteri. Immunohistochemical staining of collagenase in sections of rat uterus showed the enzyme to be present in the perinuclear region of the smooth muscle cells only of the involuting myometrium. No detectable collagenase was present in the prepartum or nonpregnant uterus. Identity of the smooth muscle cells was confirmed using an anti-smooth muscle actin antibody. In addition, the cultured uterine cells from which the immunizing antigen was obtained were also identified as smooth muscle cells. Specificity of the tissue staining was confirmed by the ability of pure rat uterine collagenase to block the reaction of the antibody with the tissue. These observations indicate that smooth muscle cells are capable of producing collagenase and are consistent with the hypothesis that this enzyme presides over the massive collagen degradation seen in postpartum uterine involution. Furthermore, measurement of collagenase bound to uterine collagen revealed that collagenase activity could be detected only at the time that the cells could be seen to be producing the enzyme by immunolocalization. These findings support the concept that collagenase is produced only as needed and not stored, either intra- or extra-cellularly.     

内分泌细胞中溶酶体对激素分泌调节的作用

Enzyme cytochemistry and immunocytochemistry were utilized to study the morphological alterations of the lysosomes and associated crinophagic and autophagic structures in the hypo-secreting pituitary gonadotrophin and Leydig cells induced by exogenous androgen. The lysosomes and autophagic vacuoles in the electron micrographs were quantitatively analysed. The morphological and quantitative data led to the following conclusions: 1) The hypo-secreting gonadotrophin showed an increase in the number of lysosomes and an enhancement of crinophagy. It demonstrated once again that the lysosomes in the protein and polypeptide hormone secreting cells play a role in the regulation of secretion process by means of the crinophagy. 2) The hypo-secreting Leydig cells showed an increase in the number of lysosomes and an enhancement of autophagic activity. This indicated that the lysosomes in the steroid hormone secreting cells also function in the regulation of hormone secretion but by means of autophagy which scavenge a part of steroid-producing apparatus and hormone. The autophagy might have similar effect in regulation of steroid secretion to the crinophagy in regulation of protein secretion.     

Preferential secretion of collagen type 3 versus type 1 from adventitial fibroblasts stimulated by TGF-β/Smad3-treated medial smooth muscle cells

Restenosis, or arterial lumen re-narrowing, occurs in 30–50% of the patients undergoing angioplasty. Adaptive remodeling is the compensatory enlargement of the vessel size, and has been reported to prevent the deleterious effects of restenosis. Our previous studies have shown that elevated transforming growth factor (TGF-β) and its signaling protein Smad3 in the media layer induce adaptive remodeling of angioplastied rat carotid artery accompanying an increase of total collagen in the adventitia. In order to gain insights into a possible role of collagen in Smad3-induced adaptive remodeling, here we have investigated a mechanism of cell–cell communication between medial smooth muscle cells (SMCs) and adventitial fibroblasts in regulating the secretion of two major collagen subtypes. We have identified a preferential collagen-3 versus collagen-1 secretion by adventitial fibroblasts following stimulation by the conditioned medium from the TGF-β1-treated/Smad3-expressing medial smooth muscle cells (SMCs), which contained higher levels of CTGF and IGF2 as compared to control medium. Treating the TGF-β/Smad3-stimulated SMCs with an siRNA to either CTGF or IGF2 reversed the effect of conditioned media on preferential collagen-3 secretion from fibroblasts. Moreover, recombinant CTGF and IGF2 together stimulated adventitial fibroblasts to preferentially secrete collagen-3 versus collagen-1. This is the first study to identify a preferential secretion of collagen-3 versus collagen-1 from adventitial fibroblasts as a result of TGF-β/Smad3 stimulation of medial SMCs, and that CTGF and IGF2 function together to mediate this signaling communication between the two cell types.     

内分泌细胞中溶酶体对激素分泌调节的作用

本实验用外源性雄激素引起垂体促性腺激素细胞和睾丸间质细胞分泌抑制,对这两种细胞中的溶酶体及分泌吞噬和自体吞噬活动进行了超微结构形态观察和半定量分析。实验中应用了CMP酶细胞化学技术和免疫胶体金技术。研究结果显示,在分泌受抑制状态下,垂体促性腺激素细胞中溶酶体增多,分泌吞噬活动加强;与此同时,睾丸间质细胞也表现溶酶体增多、自体吞噬活动加强。这些结果不仅再次证明在分泌蛋白质激素细胞中溶酶体以分泌吞噬的方式参与了激素分泌调节,更重要的是初步证明在分泌类固醇激素细胞的分泌调节中,也有溶酶体的参与,其形式是自体吞噬作用。细胞通过自体吞噬作用得以在短时间内清除一部分合成激素的细胞器和其中的激素,这可能是分泌类固醇激素的细胞及时有效地调整激素分泌量的一项重要机制,与分泌蛋白质激素细胞的分泌吞噬有着相同的意义。     

A nude mouse xenograft model of fetal intestine development and differentiation.

This report describes a novel in vivo model of intestinal differentiation. Fourteen day, undifferentiated fetal rat small intestine, stripped of the major part of its mesenchyme, suspended in a type I collagen gel and then xenografted into a nude mouse, undergoes small intestinal morphogenesis and cytodifferentiation. All four major epithelial lineages, namely Paneth, goblet, columnar and endocrine are present. Double-label nonisotopic in situ hybridization, employing biotinylated and digoxigenin-labelled whole rat DNA and whole mouse DNA probes, was performed to distinguish donor cells from host cell types. The outer longitudinal smooth muscle layer, and the major part of the lamina propria, including pericryptal fibroblasts, are of host mouse origin; the inner circular smooth muscle layer is of donor rat origin. Cells of the muscularis propria and lamina propria acquired smooth muscle alpha-actin, presumably under the influence of the donor endoderm. Furthermore, this xenograft develops a host vascular network, and cells with the morphological appearance of lymphocytes are present within the intestinal epithelium. The production of chemotactic factors by the endoderm is postulated because grafting of collagen gel alone results in a minimal invasion by stromal cells which do not express smooth muscle alpha-actin.     

Biosynthesis of chick type VI collagen. II. Processing and secretion in fibroblasts and smooth muscle cells

The biosynthesis of type VI collagen was studied in "matrix-free" chick embryo smooth muscle cells and fibroblasts. Omission of ascorbate from the culture affected to a great extent the secretion in fibroblasts but had a very minor effect on smooth muscle cells. Quantitative analysis of the secretion process in continuous time course and in pulse-chase experiments confirmed that fibroblasts and smooth muscle cells secreted type VI collagen with the same chain composition but with different kinetics: after 4 h of chase more than 60% of the labeled type VI collagen was present in the culture medium of fibroblasts, whereas at the same time interval less than 25% was secreted by smooth muscle cells. The different kinetics depends on intrinsic properties of the cells, since it was detected also in adherent cells. However, even in fibroblasts, secretion of type VI collagen was much slower than secretion of fibronectin, of which more than 50% was already in the cell medium after 1 h of chase. Treatment of the cells with inhibitors of hydroxylation and glycosylation caused a shift in mobility that revealed a size heterogeneity in the Mr = 260,000 subunit. No evidence of processing was observed in chick cells for any of the subunits that were synthesized and secreted uncleaved. In addition, after several days of chase the Mr of the subunits of type VI collagen isolated from the matrix remained unchanged, thus excluding that in the chick even a partial or incomplete processing takes place.     

An analysis of the interactions between sympathetic nerve fibers and smooth muscle cells in tissue culture

The interactions between sympathetic nerve fibers and smooth muscle cells and fibroblasts from the newborn guinea pig vas deferens were studied in tissue culture with phase contrast microscopy, time-lapse microcinematography, catecholamine fluorescence histochemistry and scanning and transmission electron microscopy. The amount of sympathetic nerve fiber growth, its catecholamine fluorescence reaction and the size of the nerve cell bodies and their nuclei all increased in the presence of vas deferens tissue. Specific growth of nerve fibers to large clumps of vas deferens tissue was seen from distances of up to 2 mm. In contrast, no specific growth from a distance occurred to single cells or small groups of cells. However, random contact with a muscle cell often led to close, extensive, and long-lasting associations. Contact with fibroblasts was always transitory.The rate of sympathetic nerve fiber growth over individual muscle cells was faster than over fibroblasts, which, in turn, was faster than over the collagen-coated surface of the coverslip. Palpation of a muscle cell by a nerve fiber growth cone increased the rate of spontaneous contraction of the muscle cell, the extent of the increase being dependent on the number of nerve fibers involved. Multiple innervation of a smooth muscle cell occurred if nerve fibers reached the cell at about the same time, but not if there was a close association already established. These results are discussed in relation to possible interactions of sympathetic nerve fibers with smooth muscle cells in vivo.     

Granulolysis in neurosecretory neurons of the rat supraoptico-posthypophyseal system

Ultrastructural and cytochemical observations on neurosecretory neurons of the rat supraoptico-posthypophyseal systems were made under experimental conditions which resulted in striking changes in the amount of neurosecretory granules and lysosomes. Attention was focused on granulolysis. At the onset of rehydration following a 4 days water deprivation, very active autophagy took place in neurosecretory axons of the neural lobe involving the marked increase in smooth endoplasmic reticulum, microvesicles and neurosecretory granules, although the latter were still very few due to previous depletion. When axonal transport was inhibited by colchicine at the onset of rehydration, granules accumulated in the perikarya while granule reloading of the neural lobe was delayed. However autophagy, although always active in axons, remained scarce in perikarya. Moreover, in the latter there was only slight evidence of crinophagy. Hypophysectomy also induced granule accumulation in the perikarya, although accompanied by little granulolysis. Images indicative of crinophagy as shown by acid phosphatase localization were few and exclusively restricted to perikarya, while autophagy occurred essentially in axons. Autophagy appeared to be the predominant process for granulolysis and might be considered here as an aspect of the general turnover of cell constituents, related to the sudden regression of hyperactivity-induced hyperthrophy, rather than as an expression of a specific regulation of an excess of secretory material.     

Isolation and culture of rat superior mesenteric artery smooth muscle cells

Summary The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.     

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

Background

The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution.

Methods

We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20).

Results

We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2.

Conclusion

These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.     

Phagocytosis of cytoplasmic blebs derived from smooth muscle cells by fibroblast-like cells and macrophages in the post-partum rat uterus

Numerous blebs were observed in contact with smooth muscle cells (SMC) by light microscopy in the myometrium of the rat uterus after parturition. Electron-microscopically the cell surface of SMC showed bulbous protrusions, which often lacked a basement membrane and were less electron-dense than the surrounding cytoplasm or sometimes nearly electron-lucent. Many bulbous protrusions were separated from SMC and became the isolated structures which we called cytoplasmic blebs. These bulbous protrusions and cytoplasmic blebs were often found to be phagocytosed by fibroblast-like cells and macrophages. A series of these tissue changes in the uterine myometrium after delivery, possibly due to hypoxic conditions, contribute to a rapid involution of SMC which have enlarged during pregnancy.     

Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus

The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.     

Use of the p-nitrophenyl phosphate method for the demonstration of acid phosphatase during starvation and cell autolysis in the planarian Polycelis tenuis Iijima.

Synopsis Acid phosphatase activity is demonstrated employingp-nitrophenyl phosphate as substrate and lead acetate as coupler. The fine structural localization of the enzyme in starved planarian tissues is described. The method is used to pin-point starvation-induced acid phosphatase activity in relation to autophagy and crinophagy in the gland cells; autophagy, autolysis and cell death in parenchymal and gastrodermal cells and basement membrane lysis. Attention is also payed to the demonstration of muscle lysis. The histochemical implications of the method are discussed.     

Dense-cored membrane structures in rat myometrial smooth muscle cells: increase in number during parturition

Membrane-bound structures containing electron-dense material were found in the cytoplasm of the smooth muscle cells of rat myometrium. Most of these structures were observed as membrane-bound vesicles with a variable diameter in the range of 50-90 nm. Some of these were present underneath the cell membranes among the clusters of caveolae and some were found deep in the interior of the smooth muscle cell. The number of such structures progressively increased with gestation reaching the maximum during parturition and decreasing to a minimal level 2 days postpartum, suggesting that the occurrence of these structures is of physiological relevance in parturition. Ovariectomy of the pregnant rats had no influence upon the temporal distribution of these structures within the gestation period, indicating that hormones of ovarian origin are not responsible for the maintenance of these structures during their disappearance after parturition.     

Electron microscopic and cytochemical studies of rat aorta. Intracellular vesicles containing elastin- and collagen-like material

Synopsis Small, rounded vesicles with a dense core of amorphous material were observed in all cell types in the young rat aorta, that is, endothelial cells, smooth muscle cells and fibroblasts. They were particularly numerous in the Golgi complex but were also found in the cell periphery. The content of the vesicles had staining characteristics identical to those of elastin. Material of the same type was also found in cisternae on the maturing side of the dictyosomes and in vesicles budding from them. Reaction product for thiamine pyrophosphatase was present in both these structures, indicating that the Golgi complex is responsible for the formation of the dense-cored vesicles. This was further supported by the absence of reaction product for acid phosphatase in the cisternae and in the vesicles. Moreover, no uptake of exogenous markers was noted in the latter. On the basis of these findings it is suggested that the dense-cored vesicles have a secretory function and contain precursors of elastin.Elongated vesicles or profiles containing collagen fibrils were observed in smooth muscle cells and fibroblasts. In the cell periphery, these vesicles were often found to communicate with the extracellular space. Further inside the cells, they showed a close spatial relationship to the Golgi complex. Neither thiamine pyrophosphatase nor acid phosphatase activity was demonstrated in the elongated vesicles. Like the plasma membrane, their limiting membrane was positively stained for alkaline phosphatase. On the basis of these findings and the absence of uptake of exogenous markers in them, it is suggested that the elongated vesicles represent a means for collagen secretion in the growing aortic wall. The Golgi complex is believed to be involved in the transfer of collagen to these vesicles.     

Are gap junctions necessary for cell-to-cell coupling of smooth muscle?: An update.

Earlier, it was questioned whether gap junctions (GJs) were necessary for cell-cell communication in smooth muscle, and GJs were not seen in some smooth muscles. We reexamined this question in the myometrium and in intestinal smooth muscle, in light of current knowledge of the presence and function of GJs. In the uterus, numerous studies show that an increase in GJ number is associated with the onset of delivery and is required for effective parturition. In all cases, this increase in GJ number and the changes in uterine contractility were correlated with increased electrical and metabolic coupling. Evidence for the much smaller, but detectable, degree of electrical coupling in the preterm uterus is explained by the small (but again detectable) number of GJs present. In the intestine, GJs are readily detected in the circular muscle layer but have not been described in the adjacent longitudinal layer. While our immunohistochemical studies failed to detect GJs in the longitudinal layer, this may not be adequate to prove their absence. Therefore, current knowledge of GJ number and function is adequate to explain cell-cell coupling in the uterus. Although it remains uncertain whether GJs are absent from the longitudinal muscle of the intestine, there is no definitive evidence that cell-cell coupling can occur by means other than GJs.     

Differences in cell division and thymidine incorporation with rat and primate fibroblasts in collagen lattices.

Human and gorilla dermal fibroblasts, primate cells, suspended in a collagen lattice, do not divide for the first 3 days. In contrast, rat fibroblasts divide within 24 hr. In this study, the proliferation of rat fibroblasts were compared to primate fibroblasts. Rat fibroblasts in monolayer culture increase from 100,000 to 355,000 in 2 days, and human cells increase from 100,000 to 436,000 in the same period. An initial seeding of 100,000 rat fibroblasts suspended in collagen increased to 163,000 cells in 2 days. An initial 100,000 human fibroblasts seeded in collagen decreased to 80,000 cells in 2 days. Retarded proliferation of human and gorilla fibroblasts in collagen is unrelated to a defect in DNA synthesis. By autoradiography human fibroblasts suspended in collagen incorporate labelled thymidine. By flow cytometry analysis, the DNA concentrations of human fibroblasts suspended in collagen exhibited 41% in a 4N chromosome state, compared to 14% in monolayer culture. Nuclei of gorilla fibroblasts from collagen displayed 42% in a 4N state, compared to 19% in monolayer culture. With nuclei of rat fibroblasts from collagen, 14% were in a 4N state, compared to 9% in monolayer culture. Primate fibroblasts show a three-fold increase in the number of nuclei in a 4N state compared to rat fibroblasts suspended in collagen. After replating fibroblasts released from collagen in monolayer culture in the presence of 1 mM hydroxyurea (an inhibitor of DNA synthesis) primate fibroblasts doubled in 24 hr. Under identical conditions, rat fibroblasts showed no cell division.(ABSTRACT TRUNCATED AT 250 WORDS)