Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: II. Characterization of the Phosphate Starvation Inducible-Excreted Acid Phosphatase

Three-day-old suspension cultured cells of Lycopersicon esculentum transferred to a Pi-depleted medium had 2.7 times the excreted acid phosphatase (Apase) activity of cells transferred to a Pi-sufficient medium. Cell growth during this time period was identical for the two treatments. Excreted Apase activity was resolved into two fractions on a Sephadex G-150 column. Most of the phosphate starvation inducible (psi) enhancement in activity was in the lower molecular weight fraction. These two fractions exhibited different substrate versus pH activity profiles. With a native polyacrylamide gel electrophoresis assay, the lower molecular weight fraction resolved into two bands of activity. Both column fractions resolved into the same single band of activity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of this enzyme was 57 kilodalton. These data indicate that L. esculentum has at least two isozymes of the psi-excreted Apase and that these isozymes may associate to form high molecular weight aggregates. Labeling studies using [³⁵S]methionine show that the psi response in tomato cells is complex and involves changes in the steady state levels of several excreted proteins.     

Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila'', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila'' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.     

Analysis of human T cell responses to group A streptococci using fractionated Streptococcus pyogenes proteins

Cell extract and spent culture supernatant proteins from Streptococcus pyogenes Manfredo strain (type M5) were each separated to give 22 narrow range molecular weight fractions by blot-elution from SDS-polyacrylamide gels. Eluted samples and unfractionated proteins were screened for T cell stimulatory activity using human peripheral blood mononuclear cells (PBMC) from healthy adults in proliferation assays. Responses were measured in 4- and 7d cultures. Responses to a wide range of cell extract proteins were revealed by fractionation, the degree of response to each fraction varying between donors. Unfractionated culture supernatant proteins elicited proliferative responses by PBMC from all individuals examined. Responses to culture supernatant fractions containing 25–33 kDa proteins could be attributed to known superantigens. Furthermore, samples from culture supernatants containing higher molecular weight fractions (>45 kDa) elicited responses in 50% of donors in 7d cultures, suggesting that these fractions contained common recall antigens. The efficacy of using electroeluted samples to identify T lymphocyte stimulatory proteins was confirmed by demonstrating that a known superantigen of S. pyogenes Manfredo strain, streptococcal pyrogenic exotoxin C (SPEC), could be fractionated successfully using this method and its activity recovered. Our results show that human T cell responses to group A streptococci involve a remarkably wide range of both cell-associated and released streptococcal proteins.     

Vitamin B12 enhances the stimulation of DNA synthesis by serum in resting cultures of 3T6 cells

When low levels of serum are used to stimulate resting cultures of 3T6 cells the limiting factor in increasing DNA synthesis is a low molecular weight component, rather than a polypeptide growth factor. The former component is absent from Dulbecco's modified Eagle's medium (DEM) but present in Waymouth medium. We identify the essential component as vitamin B₁₂. Addition of vitamin B₁₂ to DEM increases the effectiveness of low levels of serum, as assessed both by [³H]TdR incorporation and autoradiography. Thus cultures of 3T6 cells in vitamin B₁₂-supplemented DEM offer a useful in vitro system for testing the mitogenic potency of polypeptides and serum fractions.     

Effects of cyclic AMP and Sephadex fractions of chick embryo extract on cloned retinal pigmented epithelium in tissue culture

The effects of dibutyryl cyclic 3',5'-adenosine monophosphate (BcAMP) and Sephadex G-25 fractions of chick embryo extract on the growth rate, morphology, and pigmentation of normal chick retinal pigmented epithelium (PE) were investigated. Seven cloned PE cell lines were each grown in modified Ham's F-12 medium alone (F-12), or in F-12 supplemented with either high molecular weight (H) or low molecular weight (L) fractions of chick embryo extract. Cells grown in F-12 alone or in L medium formed compact epithelial sheets, whereas cells grown in H had a fibrocytic appearance and formed poorly organized monolayers. In H plus BcAMP, cell morphology was more epithelioid than in H alone, and generally the monolayers appeared more differentiated. Under each of these three culture conditions, 2 x 10^-4 M BCAMP retarded the increase in cell number and decreased the final number of cells per culture dish, but had little effect on plating efficiency. BcAMP also increased the rate of cell adhesion to a plastic substratum. Pigmentation was marked in cultures grown in F-12 or in L alone, but the addition of BcAMP dramatically reduced visible pigmentation. This effect was reversed when BcAMP was removed from the culture medium. Thus BcAMP modifies cell and colonial morphology, rate of cell accumulation, adhesive properties, and pigmentation of normal PE cells.     

Uptake of bacterial DNA by Chlamydomonas reinhardi

Escherichia coli [³H]DNA supplied to vegetative cultures of wild-type (mt+) and CW15 (mt+; mutant lacking the cell wall) Chlamydomonas reinhardi could bind to the cell wall of the wild-type and to the cell membrane of CW15 mutant cells. The extent of this binding decreased with time and was to a large degree (over 90%) DNA-ase-sensitive. Nevertheless, about 0.01% of the bacterial DNA remained irreversibly associated with the cells when they reached stationary phase. The irreversible binding of the donor bacterial DNA to Chlamydomonas cells could be increased by treatment of the cultures with polycations such as DEAE-dextran, poly-L-lysine and poly-L-ornithine. Although the CW15 cells rapidly degraded bacterial DNA in the culture medium wild-type cells showed only a small effect on the molecular weight of the donor DNA.The acid-insoluble radioactivity irreversibly bound to WT (+) cells consisted mainly of oligonucleotides with a small proportion present as less depolymerized donor DNA. No radioactivity, however, was found to be associated with the recipient high molecular weight Chlamydomonas DNA.No labeled donor DNA could be recognized in the cells given bacterial [³H]DNA in early stationary phase. Instead, radioactivity found in Chlamydomonas DNA corresponded to reutilization of [³H]thymine derivatives released as a result of [³H] DNA degradation. No evidence for the integration of detectable amounts of donor DNA sequences into the host cell DNA was obtained.     

Copper accumulation and metabolism in primary monolayer cultures of rat liver parenchymal cells

The characteristics of hepatic copper accumulation and metabolism were studied using primary monolayer cultures of adult rat liver parenchymal cells. Accumulation of copper from serum-free medium was temperature dependent and strongly inhibited by cyanide and N-ethylmaleimide. Addition of various concentrations of zinc to the medium did not alter copper accumulation by the cells. Furthermore, it was found that supplementation of the cell cultures with dexamethasone significantly stimulated zinc accumulation without affecting the accumulation of copper. Cycloheximide substantially stimulated accumulation of copper from the culture medium, whereas actinomycin D had no effect. Efflux experiments showed that copper is rapidly sequestered by intracellular components and becomes unavailable for exchange soon after it is transported into the cells. Gel chromatography of liver cytosol demonstrated that most of the cooper that is initially accumulated is bound to the low molecular weight cytoplasmic protein metallothionein.     

Protein metabolism and electrophoretic profiles in astroglial primary cultures from different regions of newborn rat brain

Primary astroglial cultures (14 days of age) from cerebral cortex, striatum, and hippocampus of newborn rat brain contained similar amounts of soluble proteins. Uptake and incorporation of [³H]valine into soluble protein measured after 30 and 60 min of incubation, respectively, was on a similar level in the various cultures. [³H]valine labeling of protein bands from the cell soluble fractions and incubation media of hemisphere cultures, and which were separated by isoelectric focusing (IEF) or sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), indicated that proteins are released to the extracellular medium after being synthesized within cultivated cells. Acidic and high molecular weight proteins were more heavily labelled in the incubation media than in the cell soluble fractions. Two-dimensional electrophoresis (IEF×SDS-PAGE) of soluble proteins from the different cultures showed similar patterns, which were quite different from the serum-free extracellular protein patterns. Both fractions were different from the pattern obtained from fetal calf-serum. In striatum and hippocapus culture media a spot was seen with Ip 6.0–6.8 and m.w. 105,000, and in the media from cerebellar cultures another spot was observed with Ip 5.2–5.6 and m.w. 55,000. The results show that the different cultures are similar in their protein synthetic capacity and protein composition. The specific differences observed in proteins obtained from the serum-free incubation media might indicate specific properties among astroglial cells from various brain regions.     

Light-induced changes of enzyme activities in parsley cell suspension cultures: Increased rate of synthesis of phenylalanine ammonia-lyase

When dark-grown cell suspension cultures of parsley (Petroselinum hortense) were illuminated for increasing periods of time, increasing amounts of phenylalanine ammonialyase activity were obtained 5 hr after the onset of light.Pulses of [³⁵S]methionine of varying duration from 1 to 150 min were given to cell cultures in the dark period subsequent to a light period of 2.5 hr. The cells were harvested 5 hr after the onset of light. Analysis of the soluble proteins by polyacrylamide gel electrophoresis revealed a distinct peak of radioactivity coinciding with the activity of phenylalanine ammonia-lyase. The results of experiments in which radioactive methionine was administered for 10 min to dark-grown or light-induced cells at different times after the light period were compared. An efficient incorporation of radioactivity into the fractions possessing the enzyme activity was observed 5 hr after induction, while no significant labeling was detected either after 1.5 or 25 hr, or in extracts from nonilluminated cells. The radioactive fractions containing the enzyme activity were further analyzed by sodium dodecyl sulfate-disc gel electrophoresis. Significant amounts of radioactivity at the molecular weight of the subunits of phenylalanine ammonia-lyase (84,000) were found only in the extracts from cells which had been labeled 5 hr after induction. These results suggest that the light-induced increase in phenylalanine ammonia-lyase activity is due to de novo synthesis, but not to an activation of preformed, inactive enzyme.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Seed Dormancy in Acer: An assessment of the Rôle of the Structures Covering the Embryo

Cells of a seven year old strain of Papaver somniferum L. when cultured for 2 weeks and incubated with substances known to elicit the formation of phytoalexins, responded by turning reddish brown within 6 h and accumulating sanguinarine. Morphinan alkaloids were not detected. Media (100 ml) containing 1 ml of Botrytis spec. preparations raised the level of sanguinarine in the cells 26 times over the maximum level found in controls. Over a culture period of 79 h the cells achieved a sanguinarine concentration of 2.9% of dry weight. Media (100 ml) with 1 ml of Rhodotorula rubra preparation, 15 mg arachidonic acid, 1 mg actinomycin, 0.5 ml of Helminthosporium gramineum, Sclerotinia sclerotiorum, or 5 ml Colletotrichum gloeosporoides preparation elicited a considerable, but relatively weaker response. Sanguinarine accumulation was also found to occur in the medium and reached a concentration of 43% of total sanguinarine per culture when cells were cultured in 100 ml medium with 5 ml Colletotrichum preparation for 24 h. Young poppy cell cultures initiated 9 months ago responded to the presence of Botrytis material as did 7-year-old cultures.     

SOX2 and OCT4 mRNA-Expressing Cells,Detected by Molecular Beacons,Localize to the Center of Neurospheres during Differentiation

Neurospheres are used as in vitro assay to measure the properties of neural stem cells. To investigate the molecular and phenotypic heterogeneity of neurospheres, molecular beacons (MBs) targeted against the stem cell markers OCT4 and SOX2 were designed, and synthesized with a 2’-O-methyl RNA backbone. OCT4 and SOX2 MBs were transfected into human embryonic mesencephalon derived cells, which spontaneously form neurospheres when grown on poly-L-ornitine/fibronectin matrix and medium complemented with bFGF. OCT4 and SOX2 gene expression were tracked in individual cell using the MBs. Quantitative image analysis every day for seven days showed that the OCT4 and SOX2 mRNA-expressing cells clustered in the centre of the neurospheres cultured in differentiation medium. By contrast, cells at the periphery of the differentiating spheres developed neurite outgrowths and expressed the tyrosine hydroxylase protein, indicating terminal differentiation. Neurospheres cultured in growth medium contained OCT4 and SOX2-positive cells distributed throughout the entire sphere, and no differentiating neurones. Gene expression of SOX2 and OCT4 mRNA detected by MBs correlated well with gene and protein expression measured by qRT-PCR and immunostaining, respectively. These experimental data support the theoretical model that stem cells cluster in the centre of neurospheres, and demonstrate the use of MBs for the spatial localization of specific gene-expressing cells within heterogeneous cell populations.     

Selection, Isolation, and Characterization of Cadmium-Resistant Datura innoxia Suspension Cultures

Datura innoxia cells from suspension cultures were selected for their ability to grow and divide rapidly in normally lethal concentrations of cadmium. Cells resistant to 12.5, 25, 50, 100, 160, 200, and 250 micromolar cadmium chloride were isolated and utilized to initiate cell suspension cultures resistant to this toxic metal ion. Variant cell lines retained their ability to grow in cadmium after being grown in its absence for more than 400 generations. Resistance to cadmium was correlated with the synthesis of low molecular weight, cysteine-rich, cadium-binding proteins. Synthesis of these proteins was induced rapidly in cadmium-resistant cells in response to a challenge of cadmium. Induction was detectable within one hour after exposure of the cells to the metal ion. Accumulation of protein bound cadmium reached a maximum eight to twelve hours following exposure. Metal-binding proteins were not detectable in the cadmium sensitive D. innoxia cells from which resistant cells were derived.     

The heparan sulfates of Swiss mouse 3T3 cells. The effect of transformation

Three major pools of heparan sulfate have been isolated from cultures of Swiss mouse 3T3 and SV40-transformed 3T3 cells: cell-surface, medium, and intracellular heparan sulfates. The cell-surface heparan sulfate is a high molecular weight proteogylcan which is partially degraded by pronase. Before pronase treatment, it has a peak molecular weight (as estimated by gel filtration) of appox. 7.2 · 10⁵ in contrast to only 2.4 · 10⁵ after pronase treatment. The medium heparan sulfate appears to be similar in structure to the cell-surface heparan sulfate, since they coelute on Bio-Gel A-15m and DEAE-cellulose, and are both proteoglycans. In contrast, the intracellular heparan sulfate has a low molecular weight (6.0 · 10³) and has little if any attached protein. Both the medium and intracellular heparan sulfate exhibit the transformation-associated change in structure reported earlier for cell-surface heparan sulfate (Underhill, C.B. and Keller, J.M. (1975) Biochem. Biophys. Res. Commun. 63, 448–454). This transformation-associated change, detected by DEAE-cellulose chromatography is not the result of changes in either molecular weight or protein core. Cellulose acetate electrophoresis of the cell-surface heparan sulfate at pH 1 suggests that the transformation-associated change in structure is due to a difference in sulfate content. Both types of heparan sulfate are produced in mixed cultures ot 3T3 and SV3T3 cells, indicating that neither serum factors in the culture medium nor secreted cell products are responsible for the transformation-associated change in heparan sulfate structure. The presented date are discussed with respect to the postulated role of heparan sulfate in cell social behavior.     

Specific biochemical and immunological properties of some water-soluble glycoproteins produced by rat gastric mucosal scrapings in vitro

• 1.1. Two major glycoprotein components were released in vitro by rat gastric mucosal cells after 4 hr incubation with radioisotopes: a high molecular weight fraction with the characteristics of a fucomucin and a low molecular weight fraction, the latter having a higher specific radioactivity than the former.
• 2.2. Pulse chase experiments indicate that several low and high molecular weight glycoproteins are synthesized simultaneously with no precursor-product relationship between them.
• 3.3. Common antigenic determinants, specific to the stomach were found on the 2 fractions, using immunofluorescence, both fractions appeared to be present in the same cells.

     

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.     

Use of β-cyclodextrins to enhance phytosterol production in cell suspension cultures of carrot (Daucus carota L.)

Phytosterols are isoprenoid-derived compounds that play essential roles in plant growth and development as they are integral components of the plant cell membranes, and responsible for their permeability and fluidity. In this study, the effect of different types of beta-cyclodextrins (β-CD) on phytosterol production was evaluated using Daucus carota cell suspension cultures. A detailed analysis provides the optimal type and concentration of β-CD, elicitation time, and the optimal cell age and density. The highest levels of phytosterols produced by cells and secreted to the culture medium were obtained when 10 day-old D. carota cell suspension cultures, with an initial cell density of 200 g of fresh weight (FW) l^?1, were incubated in the presence of 50 mM of methylated-β-CD (M-β-CD) for 144 h. In addition, M-β-CD significantly promoted the accumulation of phytosterol in the extracellular medium of D. carota cell suspension cultures, which was not observed in control cell suspension cultures. Moreover, these high phytosterol levels did not improve when methyl jasmonate (MJ) was added to the cell suspension cultures alone or combined with 50 mM M-β-CD, although MJ stimulated the formation of defense-related compounds. Therefore, the use of carrot cell suspension cultures seems a promising biotechnological alternative since the addition of β-CD to the culture medium not only induced the biosynthesis of phytosterols but also promoted their secretion into the culture medium, where they were accumulated and could be isolated easily.     

Isolation of a kaurene synthetase inhibitor from castor bean seedlings and cell suspension cultures

Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures and seedlings of castor bean. Both cell-free extracts contain an inhibitor of kaurene synthetase. The inhibition affects mainly the cyclization of geranylgeranyl pyrophosphate to copalyl pyrophosphate (activity A) and has little or no effect on the further cyclization of copalyl pyrophosphate to ent-kaurene (activity B) in both castor bean and Fusarium moniliforme cell-free enzyme preparations. In castor bean cell suspension cultures, the inhibitor diffuses out of the cells to the growth medium. The inhibitor is stable to 100 C heat treatment for 10 minutes and exposure to pH values of 2.0 or 13.0, and it diffuses through a dialysis bag (10⁴-dalton cutoff). Gel filtration chromatography of the inhibitor on a calibrated Bio-Gel P-10 column indicated a molecular weight of 7,500. Kinetic studies indicate that the inhibition of activity of A of kaurene synthetase is noncompetitive and reversible.     

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.