Temperature-sensitive cell cycle mutants: a chinese hamster cell line with a reversible block in cytokinesis.

A thermosensitive line (TS 111) was isolated from a suspension culture of Chinese hamster fibroblasts, using a BUdR suicide selection technique. In this line, cytokinesis is blocked at 39 degrees C. DNA and protein synthesis are not arrested but keep on at a steady rate. Giant cells are produced which accumulate either numerous nuclei or one big nucleus with several nucleoli and more than a hundred chromosomes. At each nuclear cycle, all the chromosomes in the cell appear to condense in a synchronous manner, although it is possible that not all the sets of chromosomes duplicate. When the culture is returned to the permissive temperature (34 degrees C) after a prolonged arrest at the restrictive temperature, cytokinesis resumes with early extrusion of karyoplasts from multinucleated cells. The division block is independent of cell density in suspension culture and is not prevented by cell contact when cells grow attached to Petri dishes. At 34 degrees C, a residual expression of the mutation is indicated by the presence of binucleate and up to 30% anucleate cells. A remarkable similarity and some synergism exists between the mutation and cytochalasin B effects.     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.     

Phosphoinositide-specific phospholipase C of a cloned mast cell line, mastocytoma P-815: purification and some characterization

1. Multiple forms of phosphoinositide-phospholipase C (PLC) were isolated from mastocytoma; two cytosolic forms (cPLC-I, Mr 150,000; cPLC-II, Mr 110,000) and two membrane-associated forms (mPLC-I, Mr 85,000; mPLC-II, Mr 85,000). 2. Four PLC forms differently behaved in substrate specificity and effect of GTP-binding proteins.     

Evidence for an intraclonal random shift between two types of cell cycle times in an embryonal carcinoma cell line

In a recent paper we reported the discovery of an intraclonal bimodal-like cell cycle time variation within the multipotent embryonal carcinoma (EC) PCC3 N/1 line growing in the exponential phase in the undifferentiated state. The variability was found to be localized in the G1 period. Furthermore, an inverse relation between cell size and cell generation time was found in the cell system analysed. It was suggested that the bimodal-like intraclonal time variability previously reported was attributable to an intraclonal shift between two types of cell-growth-rate cycles and that the cell-growth cycle has a supramitotic character, being dissociated from the DNA-division cycle. The growth rate heterogeneity in the cell population was found to need three cell cycles to reach full dispersion in time. This was assumed to be due to a decreased inheritance from sister cell pairs to second cousin cell pairs. Thus, the interesting feature is that in one and the same multipotent cell line there was evidence for an intraclonal instability with a random shift between two types of cell cycle differing in the duration of their G1 period.     

Computer modelling and experimental evidence for two steady states in the photosynthetic Calvin cycle.

We present observations of photosynthetic carbon dioxide assimilation, and leaf starch content from genetically modified tobacco (Nicotiana tabacum) plants in which the activity of the Calvin cycle enzyme, sedoheptulose-1,7-bisphosphatase, is reduced by an antisense construct. The measurements were made on leaves of varying ages and used to calculate the flux control coefficients of sedoheptulose-1,7-bisphosphatase over photosynthetic assimilation and starch synthesis. These calculations suggest that control coefficients for both are negative in young leaves, and positive in mature leaves. This behaviour is compared to control coefficients obtained from a detailed computer model of the Calvin cycle. The comparison demonstrates that the experimental observations are consistent with bistable behaviour exhibited by the model, and provides the first experimental evidence that such behaviour in the Calvin cycle occurs in vivo as well as in silico.     

Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of an adriamycin binding site in the secretory granules of the mast cell

Adriamycin induced significant non-cytotoxic histamine release from rat peritoneal mast cells to which the drug showed a very high affinity. The relationship between adriamycin-induced exocytosis and its uptake by purified rat peritoneal mast cells was studied. Adriamycin induced histamine release and was highly concentrated in mast cells at 37 degrees C but not at 0 degrees C. However, if exocytosis was provoked by other secretagogues like compound 48/80, protamine, concanavalin A, and ionophore A23187, and cells were then treated with adriamycin at 0 degrees C, the concentrations of the antineoplastic drug significantly increased. Adriamycin binding to purified granular material was similar to that of intact cells treated at 37 degrees C, but was not modified by metabolic inhibitors, extremes of temperature (0 or 45 degrees C) or by the carboxylic ionophore monensin. On the contrary, sodium cromoglycate limited adriamycin binding to granular materials as well. In addition, sodium cromoglycate, but not monensin, displaced the antineoplastic drug from mast cells, even when added after adriamycin. We conclude that the high affinity of adriamycin for mast cells is ascribable to the externalization of a granular binding site, as a consequence of the exocytotic process. The experiments with sodium cromoglycate suggest that this binding site could be in common with the antiallergic drug.     

Identification of two different states of P-glycoprotein in its catalytic cycle: role of the linker region in the transition between these two states

P-glycoprotein (Pgp) is a drug-translocating ATPase responsible for multidrug resistance in cancer. Although it is well-established that Pgp exhibits drug-dependent ATPase and ATP-dependent drug transport functions, the mechanism by which these two reactions are coupled remains unclear. We have shown recently that proteolytic cleavage of the linker region, which joins the NH2 and COOH halves of the Pgp molecule, results in a Pgp form that exhibits drug-independent and -dependent ATPase activities (Nuti et al., (2000) Biochemistry 39, 3424-3432; Nuti, S. L., and Rao, U. S. (2002) J. Biol. Chem. 277, 29417-29423). To understand the mechanism underlying this phenomenon, we used the procedure of vanadate-mediated trapping of the Pgp transport cycle intermediates to determine the steps in the catalytic cycle that are being regulated by the linker region. We show that vanadate stably traps Pgp under two different conditions, one in the presence of ATP alone and the other in the presence of ATP and drug, suggesting the existence of two Pgp conformations. These two conformations, one mediating basal and the other drug-stimulated ATPase reactions, represent different transport cycle intermediates of Pgp, because arresting Pgp in either conformation prevents the catalytic cycle from proceeding to completion. The results also show that these two conformations are uncoupled and appear simultaneously in Pgp that was cleaved in the linker region. These results together suggest that Pgp assumes at least two distinct conformational states, which catalyze two ATP hydrolysis events in the drug transport cycle, and the linker region mediates the transition between these two states of Pgp.     

Fluorescence of mast cell granules in paraffin sections and cell smears induced by an N-quaternary oxazole scintillator

Summary The N-quaternized derivative of dimethyl-POPOP (termed Q4) induces a bluish-green fluorescent reaction in mast cell granules from paraffin sections and cell smears, in addition to a previously described bluish-white fluorescent reaction in chromatin DNA. The chromatin reaction was abolished by staining the samples either with Mayer's Haematoxylin before Q4 treatment or by Q4 treatment at pH 1.5. The reaction in mast cell granules was absent after substrate methylation. The staining sequence Haematoxylin-Eosin-Q4 also worked well in paraffin sections, allowing the observation of the current histological image under bright-field illumination as well as double-colour emission under fluorescence microscopy. The sequence is proposed as a new diagnostic procedure for demonstrating mast cell granules.     

Uptake of 5-Hydroxytryptamine by mast cells in vivo: A cytofluorometric study of mast cells and individual mast cell granules

Summary Uptake, distribution and turnover of 5-Hydroxytryptamine (5-HT) was studied by cytofluorometric analysis of whole mast cells and individual granules. Injection of 5-HT as well as 5-Hydroxytryptophan (5-HTP) intraperitoneally or subcutaneously resulted in a parallel uptake of 5-HT in cells and granules. Intraperitoneal injections of 5-HT in such small quantities that may be available under physiological conditions resulted in an increase in fluorescence intensity of the mast cells, indicating a very efficient uptake mechanism for 5-HT in vivo. Much larger doses of 5-HTP were required to obtain a corresponding uptake of 5-HT in the mast cells. The 5-HT was rather rapidly taken up in the granules and eliminated very slowly, at the same rate both from granules and mast cells. The low elimination rate confirms our previous findings that the turnover of 5-HT is much lower in mast cells than in other amine containing cell systems. The combination of an extremely efficient, rapid uptake of 5-HT with a slow elimination suggests a specific function for mast cells in the regulation of free amine concentrations in tissues.Supported by grants from the Swedish Medical Research Council, Project no 2235     

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.     

Lysine decarboxylase mutants of Escherichia coli: evidence for two enzyme forms

Mutants with reduced lysine decarboxylase activity were isolated from an Escherichia coli polyamine auxotroph. These mutants could not produce induced enzyme, showing only a very low level of an apparently constitutive form. Both enzyme forms could be demonstrated in the parental strain.     

Uptake of 5-hydroxytryptamine by mast cells in vivo: a cytofluorometric study of mast cells and individual mast cell granules.

Uptake, distribution and turnover of 5-Hydroxytryptamine (5-HT) was studied by cytofluorometric analysis of whole mast cells and individual granules. Injection of 5-HT as well as 5-Hydroxytryptophan (5-HTP) intraperitoneally or subcutaneously resulted in a parallel uptake of 5-HT in cells and granules. Intraperitoneal injections of 5-HT in such small quantities that may be available under physiological conditions resulted in an increase in fluorescence intensity of the mast cells, indicating a very efficient uptake mechanism for 5-HT in vivo. Much larger doses of 5-HTP were required to obtain a corresponding uptake of 5-HT in the mast cells. The 5-HT was rather rapidly taken up in the granules and eliminated very slowly, at the same rate both from granules and mast cells. The low elimination rate confirms our previous findings that the turnover of 5-HT is much lower in mast cells than in other amine containing cell systems. The combination of an extremely efficient, rapid uptake of 5-HT with a slow elimination suggests a specific function for mast cells in the regulation of free amine concentrations in tissues.     

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.     

The molecular basis for the storage and release of histamine in rat mast cell granules

The proposal that mast cell histamine is stored in a histamine-heparin or histamine-zinc-heparin complex is criticised. In the first place, both heparin and zinc are present in amounts sufficient to bind only a minor part of the histamine stored. Secondly, it is evident from titration studies on the granules that the ester sulphate groups of heparin, which are essential for the binding of histamine, are already occupied since they are involved in the binding between heparin and basic protein in the complex that forms the matrix of the basophil granules. This protein-heparin complex is the histamine-binding material. It has the properties of a weak cation exchange resin, that binds organic and inorganic cations unselectively. Histamine release from mast cells is a simple cationic exchange between histamine and inorganic cations (mainly sodium) which takes place in granules which become exposed to the extracellular medium during the exocytotic process. The possibility that other release processes are also based on ionic exchange is briefly discussed.     

Complementation between two temperature-sensitive mammalian cell mutants, each defective in the G1 phase of the cell cycle

Two mammalian temperature-sensitive (ts) G1 cell cycle mutants of different species origin (Syrian hamster and mouse) have been tested for complementation using somatic cell hybrid analysis. All hamster-mouse hybrid clones tested were found to exhibit normal growth properties at the restrictive temperature, while neither mutant alone was capable of normal growth at this temperature. The two mutant lines therefore complement for growth in a somatic cell hybrid and most likely represent ts lesions in different cellular functions specific to the G1 phase of the cell cycle.     

Adrenal chromaffin granules: evidence for an ultrastructural equivalent of the proton-pumping ATPase

Adrenal chromaffin granules are known to possess an F1-ATPase which according to biochemical criteria is very similar to the mitochondrial one. To find a morphological equivalent for this enzyme chromaffin granules from bovine adrenal medullar were subjected to negative staining and freeze-etching. With both methods globular particles of 8 to 9 min diameter could be demonstrated on the surface of these organelles. A single granule possessed on average 22 particles. In negative staining the particles appeared separated from the membrane by a stalk of 8 nm. This typical morphological appearance was independent from a great variety of experimental procedures. After freeze-etching the particles were closely apposed to the membrane without any evidence for an interposed stalk. Pretreatment of chromaffin granules with pronase or trypsin led to a time dependent disappearance of the surface particles. In negative staining the stalked of chromaffin granules were found to be very similar in structure and size to those of mitochondria which have already been identified as F1-complexes. Based on this observation and other lines of evidence we suggest that the stalk particles found on the surface of chromaffin granules represent the F1-complex of the proton-pumping ATPase of these organelles.     

Male germ line development in Arabidopsis. duo pollen mutants reveal gametophytic regulators of generative cell cycle progression

Male germ line development in flowering plants is initiated with the formation of the generative cell that is the progenitor of the two sperm cells. While structural features of the generative cell are well documented, genetic programs required for generative cell cycle progression are unknown. We describe two novel Arabidopsis (Arabidopsis thaliana) mutants, duo pollen1 (duo1) and duo pollen2 (duo2), in which generative cell division is blocked, resulting in the formation of bicellular pollen grains at anthesis. duo1 and duo2 map to different chromosomes and act gametophytically in a male-specific manner. Both duo mutants progress normally through the first haploid division at pollen mitosis I (PMI) but fail at distinct stages of the generative cell cycle. Mutant generative cells in duo1 pollen fail to enter mitosis at G2-M transition, whereas mutant generative cells in duo2 enter PMII but arrest at prometaphase. In wild-type plants, generative and sperm nuclei enter S phase soon after inception, implying that male gametic cells follow a simple S to M cycle. Mutant generative nuclei in duo1 complete DNA synthesis but bypass PMII and enter an endocycle during pollen maturation. However, mutant generative nuclei in duo2 arrest in prometaphase of PMII with a 2C DNA content. Our results identify two essential gametophytic loci required for progression through different phases of the generative cell cycle, providing the first evidence to our knowledge for genetic regulators of male germ line development in flowering plants.