Induction of granulopoiesis of mastocytoma cells: ultrastructural and cytochemical studies on production of serotonin in a cultured mouse mastocytoma cell line

Electron microscopic observations of an originally established mouse mastocytoma cell line (BSP-MST-2) revealed that the cytoplasm of many of the MST-2 cells contained small and low osmiophilic granules and a few mature electron-dense granules. Fluorescent- and immuno-histochemical examinations also suggested the immaturity of granules as the cytoplasmic reaction for serotonin (5-HT) was weak. Induction of further maturation of granules was investigated by administration of various chemical agents. Among the chemicals examined, sodium butyrate and hydrocortisone were effective. In the presence of 1 mM sodium butyrate for 24 h, the cytoplasmic granules contained an abundant dense matrix. MST-2 cells incubated with hydrocortisone at 5 micrograms/ml for 24 h showed a somewhat different granulopoietic pattern from those incubated with sodium butyrate, including numerous electron-dense progranules. Fluorescent- and immuno-histochemical studies showed increased reactions of cytoplasmic 5-HT of both butyrate- and hydrocortisone-treated MST-2 cells. The specificity of these morphological and cytochemical changes was confirmed by treatment with reserpine, a drug which depletes cellular 5-HT; electron-dense materials were virtually diminished and cytochemical reactions were significantly decreased. The mode of induced production of 5-HT in mastocytoma granules is discussed, in relation to mastocyte differentiation.     

Effect of sodium butyrate on the production of serotonin, histamine and glycosaminoglycans by cultured murine mastocytoma cells

Effect of sodium butyrate on the cellular serotonin, histamine and glycosaminoglycans (GAGs) contents of mastocytoma p-815 cells was examined. When mastocytoma p-815-4 cells were cultured for 4 days in the presence of butyrate at 2 mM, the optimal concentration for the induction of granulopoiesis in this cell line, the cellular serotonin, histamine and GAGs contents increased markedly: serotonin increased almost 7 times, histamine 140 times and total GAGs 10 times. Chondroitinase ABC-resistant GAGs, heparin and/or heparan sulfate, increased 21 times. These cellular products reached at 3 or 4 days culture their maximal levels which depended on the amount of butyrate added up to 2 mM. The effectiveness of butyrate varied among the cloned cell lines: serotonin and histamine contents did not increase in mastocytoma p-815-6 cells, in which butyrate failed to induce granulopoiesis.     

Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.     

Ultrastructural and cytochemical studies on the cytodifferentiation of duodenal endocrine cells

Summary The development and cytodifferentiation of endocrine cells that produce the gastrointestinal hormones gastrin, cholecystokinin and secretin have been studied by a combined fluorescence-cytochemical, immunocytochemical and ultrastructural approach. The results show that, during development, several ultrastructurally distinct cell types exhibit COOH-terminal gastrin and cholecystokinin immunoreactivity. Furthermore, some cells simultaneously contain both gastrin- and cholecystokinin-specific antigenic determinants. Studies on the time course of development of gastrin and cholecystokinin cells, together with the above-mentioned data, suggest that gastrin cells may be converted into cholecystokinin cells in development. During this period, gastrin, cholecystokinin and secretin cells store the biogenic monoamine, 5-hydroxytryptamine a feature not displayed by the adult counter-parts of these cells. In the adult duodenum, characteristic enterochromaffin (EC) cells store 5-hydroxytryptamin for which, evidence for a possible hormonal role has been presented. Taken together, our data indicate that the differentiation of duodenal endocrine cells occurs in distinct steps, each involving a restriction in the biosynthetic repertoire of the cell.     

Ultrastructural and biochemical characterization of a cloned mouse keratinocyte cell line

A cloned population of mouse C3H/He keratinocytes was obtained from the 14th passage of an epidermal cell line. A two-step cloning procedure using Petriperm dishes was performed. The cloned population, grown at 34 °C, was subcultured more than 30 times over a one year period. By day 14, three cell layers were formed; the ultrastructural morphology and immunofluorescence characterization of these layers showed numerous tonofilament bundles and well organized desmosome tonofilament structures. They thereby resemble the proliferative compartment of the epidermis. High resolution acrylamide gel electrophoresis of the keratins extracted from the cloned cells showed the presence of many keratin subunits. The tonofilaments extracted from the cell layers, as well as from the supernatant cells, contained a small quantity of high MW keratins (rel. MW 63 000; apparent isoelectric point 5.5–6.2). These results indicate that the cloned keratinocyte cell line had retained a certain maturation capacity in culture.     

Characterization and chromosomal mapping of a cDNA encoding tryptophan hydroxylase from a mouse mastocytoma cell line

A cDNA library was constructed from RNA prepared from P815 mouse mastocytoma cells and screened for tryptophan hydroxylase. An essentially full-length clone that recognizes a major mRNA species of 1.9 kb in mastocytoma cell lines and in pineal gland, duodenum, and brainstem of the mouse was obtained. The predicted amino acid sequence of this mouse mastocytoma clone showed 97 and 87% identity, respectively, with tryptophan hydroxylase clones isolated from rat and rabbit pineal glands, but the mouse clone contains an unusual 3-amino-acid duplication near the N-terminus and lacks a phosphorylation site. A fragment of the cDNA produced an enzymatically active protein when expressed in Escherichia coli, thus demonstrating that the catalytic domain is included in the C-terminal 380 amino acids. The mouse tryptophan hydroxylase locus, termed Tph, was mapped by Southern blot analysis of somatic cell hybrids and by an interspecific backcross to a position in the proximal half of chromosome 7. Because TPH has been mapped to human chromosome 11, this assignment further defines regions of homology between these mouse and human chromosomes.     

Ultrastructural and cytochemical studies on the connective tissue of chaetognaths

The hydroskeleton plays a central role in the architecture of the trunk of the Chaetognath. Its fibrous part is composed by a ‘basement membrane’ which separates the epithelial and nervous level from the locomotory muscle and other tissues which surround the general cavity. This structure corresponds to a dense connective tissue sheath; together with the aqueous phase of the general cavity it constitutes the main part of the hydroskeleton. The axes of the lateral and caudal fins are extensions of this connective tissue; they are rich in ground substance and contain several kinds of fibrils and granules.The ‘basement membrane’ is made of a network of densely packed parallel layers of collagen fibrils which form helices which wrap around the trunk. The collagen fibrils of this connective stratum are sandwiched between two basal lamina; they are embedded in a reduced extracellular matrix whose components are closely related to the architecture of the collagen fibrils. In the core of the fin, the ground substance is very abundant and classical cross-striated collagen fibrils are not to be found. A compact fibrillar transition zone is to be noted between the dense connective stratum surrounding the body and the hyaline axis of the fins. In this zone, no crossbanded collagen fibrils are to be seen.The hydroskeleton and the fins show variations within the phylum. They could be related to speciation, and the ancestral pathway of the phylum. Furthermore these variations are related to the general problem of the evolution of the extracellular matrices and collagen molecule itself.     

H-2 antigen variants in a cultured mouse leukemia cell line

We have investigated the effect of immune selection against a single gene product on a cultured mouse Friend leukemia cell line. The clonal cell line used is heterozygous at theH-2 complex and expresses theH-2 ^d andH-2 ^b haplotypes. The genes selected against were theH-2K locus alleles. Variants were obtained after a single-step selection using either antiH-2K^b or anti-H-2K^d serum. The phenotypes of the variants obtained showed an interesting asymmetry between the two haplotypes. Selection against theH 2K ^b allele resulted in the isolation of the two expected types of variant-those that had lost only H-2K^b and those that had lost both H-2K^b and the linked H-2D^b. Selection against H-2K^d yielded, exclusively, variants that had lost both the selected antigen and the linked H-2D^d. None of the variants showed an alteration in expression of antigens intrans configuration. Karyotypic analyses of the variants revealed that all the cells had retained both copies of chromosome 17 present in the wild-type cells. The results suggest that the variants did not emerge through chromosome loss.     

Ultrastructural and cytochemical studies in the mantle of Anodonta cygnea

The large central region of the mantle of Anodonta cygnea was studied using transmission and scanning electron microscopy (TEM and SEM) for ultrastructural analysis and light microscopy (LM) and TEM for cytochemical analysis. X-ray diffraction studies of the organic matrix pellicle deposited on the inner surface of the shell were also carried out. Two groups of columnar cells presenting desmosomes on the apical region were observed in the outer epithelium. One group secretes a structural and neutral mucopolysaccharide (MPS) identified with chitin, normally excreted to form the organic matrix of the shell. Another group presents numerous cytoplasmic vesicles and constitutes the predominant cells of this epithelium. Two different types of mucous cells were found in the inner epithelium. One type secretes a faintly acid mucopolysaccharide (MPS) with sulphate groups and another type secretes an association of this polysaccharide with a neutral MPS. Staining for sulfhydryl groups (probably cysteine) was also positive, suggesting the presence of proteoglycans in these secretions. A third type of cells was also observed presenting a very different ultrastructural aspect (columnar form) without large secretion masses. They may correspond to the replacing cells in this highly secretory epithelium. Elastic fibers were found on the base of the outer epithelium and amoebocytes were observed in the interepithelial tissue.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An H-2 antigen variant, referred to as ?4 + 31 clone 1, was selected by its resistance to an anti-H-2D^d antiserum (BALB.G anti-BALB/c.H-2 ^g antiH-2 ^d ). When tested by cell-mediated cytolysis, this variant was found to be sensitive to cytolytic T lymphocytes raised in the same donor-host combination as that used in raising the antiserum. Further CML characterization of this variant, reported here, indicates that the cell line is in fact resistant to anti-H-2D^d killer cells raised in a more restricted immunization, viz. BALB.G anti-BALB/cH-2 ^db ,H-2 ^g anti-H-2 ^db . It is, however, sensitive to cytolytic cells raised in (BALB.B xBALB/c-H-2db) F¹ H-2 ^b /H-2 ^db ) against the BALB/c strain. These results suggest that the variant does not express H-2D^d itself, but probably expresses CML target antigens that are missing in theH-2 ^db mutation. This in vitro-isolated variant might thus be the complementary mutation to the in vivoobtainedH-2 ^db mutation.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An (H-2k/H-2d)F1 sarcoma cell line was subjected to immunoselection using ascites fluid from a mouse growing a hybridoma secreting an anti H-2Kk antibody.One hundred random clones were picked from the surviving population and screened by direct cytolysis using the hybridoma antibody or alloantisera against H-2Kk and H-2Dk. Fifty-nine clones were resistant to all three antisera, indicating that they no longer expressed the entire H-2k haplotype. Thirty-two were resistant to the ascites and to the anti H-2Kk alloantiserum, but sensitive to the anti H-2Dk serum, indicating that they had lost H-2Kk antigen, but retained H-2Dk. Nine clones were sensitive to the alloantisera, but resistant to the hybridoma, indicating that, though they retained the product(s) recognized by the alloantiserum against H-2Kk, they had lost the site(s) that bound the hybridoma antibody. Quantitative absorption assays using lymph-node cells from young BALB.K (H-2Kk) mice as targets show that one representative clone from the last group absorbs the anti H-2Kk activity in the alloantiserum. This implies that the sensitivity of the variant clone to the alloantiserum is not due to contaminating anti C-type virus antibodies in the serum. The possible implications of these data are discussed.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

In previous studies on the emergence of H-2 antigen loss variants from an H-2^b/H-2^d heterozygous mouse leukemia cell line, it has been shown that stable variants could be obtained after a single-step selection procedure with antibody and complement. Selection against the K-end alleles revealed an asymmetry in the types of variant obtained. This paper deals with the results of selection against theD-end alleles. Once again, an asymmetry is noticed. Selection against the H-2D^d gene product results in the isolation of stable variants that have lost H-2D^d, either alone or in combination with the linked H-2K^d antigens. Selection against H-2D^b, on the other hand, has not, so far, resulted in the isolation of any stable variants. These experiments have not demonstrated unequivocally the mechanism by which these H-2 antigen loss variants are generated. However, certain preliminary considerations suggest the possibility that these variants may not, in fact, be true genetic mutants. The alternative -that these variants arose through some nongenetic, but stable mechanism — must be considered seriously.     

H-2 Antigen variants in a cultured heterozygous mouse leukemia cell line

H-2 antigen variants, derived from a heterozygous mouse Friend leukemia cell line by selection with anti-H-2 antisera and complement, were tested for susceptibility to cell-mediated cytolysis, using T-lymphocytes directed against individual H-2 antigens. The cytotoxic cells were generated in the BALB and B10 backgrounds by a combination of in vivo and in vitro immunizations. The phenotypes of the variants inferred from the patterns of resistance or susceptibility to CML were consistent with those presumed from earlier assays using antisera. The one exception was the variant cell line which was H-2D(d-) in assays using antisera, but was H-2D(d+) by CML.