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At least 955 records · Page 53Linked to original sources
PubMed1977

Ultrastructural effects of Helminthosporium maydis race T toxin on mitochondria of corn roots and protoplasts.

Zea mays inbred W64A in Texas (T, toxin sensitive) male sterile and non-male sterile (N, toxin resistant) cytoplasms were utilized. Roots of freshly germinated seeds were treated for 15 min of 2 hr with culture filtrate from liquid grown Helminthosporium maydis Race T, or with a chloroform extractable purified fraction from the culture filtrate. In the susceptible W64A T line, toxin treatment, both crude and purified, caused swelling and loss of matrix densiy in mitochondria of root cap and vacuolated cells in the region of elongation. One hour treatment with the chloroform extractable toxin fraction caused similar effects or mitochondria of isolated leaf protoplasts. This is the first report of such rapid in vivo effects of HmT toxin on mitochondria. Difficulty in obtaining consistent preservation of meristem mitochondria precluded drawing firm conclusions concerning that region of the root. In the resistant W64A N line, protoplast and root mitochondria were unaffected by the toxin.

Drug Resistance↗
PubMed1985

The incorporation of radiolabeled polyamines and methionine into turnip yellow mosaic virus in protoplasts from infected plants.

Turnip yellow mosaic virus contains large amounts of nonexchangeable spermidine and induces an accumulation of spermidine in infected Chinese cabbage. By 7 days after inoculation, a majority of protoplasts isolated from newly emerging leaves stain with fluorescent antibody to the virus. These protoplasts contain 1-2 X 10(6) virions per cell and continue to produce virus in culture for at least 48 hr. [14C]Spermidine (10 microM) was taken up by these cells in amounts comparable to the original endogenous pool within 24 hr. However, after an initial rise, the spermidine content of the cell returned to its original level, implying considerable regulation of the endogenous pool(s). Putrescine and spermine were major products of the metabolism of exogenous spermidine. Radioactivity from exogenous [14C]spermidine was also readily incorporated into the ribonucleoprotein component(s) of the virus, where it appeared as both spermidine and spermine. The specific radioactivities of the viral polyamines were approximately twice those of spermidine and spermine extracted from the whole cell. Radioactivity from [2-14C]methionine was readily incorporated into the protein, spermidine, and spermine of the virus. Again, the specific activities of these amines were substantially higher in the virus than in the whole cell. Thus, newly formed virus contained predominantly newly synthesized spermidine and spermine. However, inhibition of spermidine synthesis by dicyclohexylamine led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virus.

Brassica↗
PubMed1983

High-efficiency transfer of DNA into eukaryotic cells by protoplast fusion.

Protoplast fusion is a highly efficient method for effecting gene transfer to cells in culture resulting in stable transformation at high frequency. A number of cell lines have been used successfully as recipients. There is no need to isolate and purify the DNA, which not only saves time and effort but eliminates steps that cause nicking or breaking of large cloned inserts. The high-frequency transformation achievable by protoplast fusion should make this procedure useful for studies on gene expression and for screening cloned genomic libraries for genes that can be expressed in recipient cells.

Animals↗
PubMed1990

Endonuclease-free, protoplast-forming enzyme preparation and its application in fungal transformation.

An endonuclease-free, protoplast-forming enzyme complex was prepared from the "snail enzyme." The purified preparation has high protoplast-forming activity comparable to the crude enzyme complex without destroying circular plasmid DNA. Furthermore, a higher transformation rate was achieved by the application of the endonuclease-free enzyme complex in both yeast and filamentous fungal vector-host systems.

Animals↗
PubMed1988

Sequential transposition of Tn916 among Staphylococcus aureus protoplasts.

Transposition of the Streptococcus faecalis conjugal tetracycline-resistance transposon Tn916 between S. aureus strains occurred when protoplasts of donor and recipient strains were regenerated together without prior fusion. Under these conditions, only Tn916 was transferred; spontaneous fusion of parental protoplasts is therefore unlikely to be responsible for Tn916 transfer. While the exact nature of this transfer remains unclear, it appears to resemble Tn916 conjugal transposition reported in S. faecalis. Evidence for sequential transpositions of Tn916 was obtained by 3-factorial transformation analyses and confirmed by DNA-DNA hybridizations. The ability of Tn916 to transpose within S. aureus and occupy diverse chromosomal sites demonstrates the value of this transposon in genetic studies of S. aureus.

Chromosome Mapping↗
PubMed1993

Why are quiescent mesophyll protoplasts from Nicotiana sylvestris able to re-enter into the cell cycle and re-initiate a mitotic activity?

Mesophyll protoplasts of Nicotiana sylvestris incubated in an adequate culture medium re-enter very rapidly into the cell cycle and divide. The transition G0/G1 is accompanied by a complete reversion of the program of gene expression. The program of the photosynthetic differentiated mesophyll cell is abolished whereas a new multipartite program of a highly stressed but ready-to-divide cell is established. Some genes encode proteins which structure suggests they may play key roles in these events. Most of the induced genes are under multiple controls: stress and/or development. Stress response and cellular re-organization might thus be closely related events that cannot be dissociated. It is probable that the re-entry of a protoplast into the cell cycle, ie the initial step of totipotency, closely depends on the coordinated activation of a set of genes that share common regulatory mechanisms.

Cell Cycle↗
PubMed1986

Tunicamycin and papulacandin B inhibit incorporation of specific mannoproteins into the wall of Candida albicans regenerating protoplasts.

Regeneration of Candida albicans protoplasts began with the formation of a chitin network which was complemented after a lag of about 60 min by the deposition of beta-glucan. Proteins were incorporated early to the growing structure, beginning with the mannoproteins which are kept in place by non-covalent bonds. Incorporation of covalently linked mannoproteins took place only after deposition of glucan. The incorporation of these mannoproteins did not occur when protoplasts were incubated with papulacandin B which inhibited glucan formation, or with tunicamycin which blocked N-glycosylation of mannoproteins. In the presence of papulacandin B, large amounts of native mannoproteins accumulated in the medium. However, in the presence of tunicamycin, the large mannoprotein material found was of smaller apparent molecular weight, suggesting that it was deficient in glycosylation. Partially regenerated walls were able to incorporate 'in vitro' non-covalently bound mannoproteins, indicating that some components of very large cellular structures such as walls are capable of being articulated by a self-assembly process.

Aminoglycosides↗
PubMed1996

Regulation of the supply of cytosolic oxaloacetate for mitochondrial metabolism via phosphoenolpyruvate carboxylase in barley leaf protoplasts. I. The effect of covalent modification on PEPC activity, pH response, and kinetic properties.

The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by covalent modification is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Extracts for studies on in vivo PEPC phosphorylation were prepared from barley leaf protoplasts by rapid filtration, fractionating the cell within less than 1 s. Measurements of in vitro PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. The relative PEPC phosphorylation state increased upon illumination and decreased upon redarkening under photorespiratory and non-photorespiratory conditions. PEPC activity measured in the presence of malate (3 mM) under photorespiratory conditions showed the same response indicating that a light-induced increase in PEPC activity and decrease in malate sensitivity is caused by an increased phosphorylation level of the PEPC protein. PEPC activity was pH dependent. At the physiological cytosolic pH, activity was suboptimal, but most sensitive towards malate inhibition and glucose 6-phosphate stimulation. The presence of malate increased the sensitivity of PEPC activity towards pH changes. The response of PEPC activity to changing pH was not affected by changes in the activation state of the enzyme. The Km (phosphoenolpyruvate, PEP) is about 1 mM. Upon illumination the Km (PEP) decrease significantly. Vmax was unaffected by the light treatment. The presence of physiological concentrations of glucose 6-phosphate decreased Km (PEP) 5- to 10-fold and increased Vmax by about 35%. The effect of glucose 6-phosphate was strongest (up to 7-fold) at subsaturating PEP concentrations stimulating PEPC activity to nearly maximal rates. The results show that an increase in PEPC phosphorylation state causes an increase in PEPC activity as well as in substrate affinity leading to an increased production of OAA in the light.

Cytosol↗
PubMed1989

Low heat resistance of Bacillus sphaericus spores correlated with high protoplast water content.

The low heat resistance (D100 = 0.554 min, z = 13.4 degrees C) of dormant lysozyme-sensitized spores of Bacillus sphaericus 9602 was correlated with a low protoplast wet density (1.305 g/ml) equivalent to a high protoplast water content (61.0%, wet weight basis). These values for these unusual spores were consistent with those correlated previously in 28 spore types of seven other species.

Bacillus↗
PubMed1990

Intergeneric protoplast fusion between xylanase producing Bacillus subtilis LYT and Corynebacterium acetoacidophilum ATCC 21476.

Hybrids between a strain of Bacillus subtilis isolated in our laboratory and having the ability to degrade xylan and other complex polysaccharides and Corynebacterium acetoacidophilum, a lysine producer, were prepared by protoplast fusion. Based on distinctive parental biochemical characteristics the fusants were grouped into 9 categories, viz. BC1 through BC9. Three of the hybrids, BC5, BC7a and BC7b, were tested for their ability to produce xylanase and lysine. Both BC7a and BC7b produced xylanase but BC5 did not, however all of them produced lysine albeit to different degrees. These results demonstrate that intergeneric gene transfer takes place through protoplast fusion between these 2 important genera of bacteria and some of the fusants inherit the useful traits of both the parents.

Bacillus subtilis↗
PubMed1991

Enhanced intraspecific protoplast fusion in yeast.

Intraspecific hybrid production from the polyethylene glycol induced fusion of yeast protoplasts was greatly increased when calcium propionate was included as the source of the requisite Ca2+. The use of calcium propionate, as opposed to the more commonly employed calcium chloride, resulted in substantially greater yields of hybrids from intraspecific fusions of protoplasts of Saccharomyces cerevisiae and Candida albicans. It is postulated that the ability of calcium propionate to enhance the fusion frequency is due to the anion binding to the etheric oxygen of PEG and potentiating the fusogenicity of the polymer.

Acetates↗
PubMed1991

Isolation of nuclei from Aspergillus nidulans protoplasts.

Intact nuclei were isolated from the protoplasts of the filamentous fungus Aspergillus nidulans. The large amounts of protoplasts required for such nuclear preparations were produced from young mycelia grown in liquid culture. For final purification of the crude nuclear fraction, a Nycodenz density-gradient centrifugation was applied. The resulting nuclei were of good purity and morphological state, as demonstrated by fluorescence microscopy and electronmicroscopy. The weight ratio of DNA:RNA:protein was 1:3.0:10.8 in the nuclear fraction.

Aspergillus nidulans↗
PubMed1984

A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis.

Incorporation of starch or casein into protoplast-regeneration medium facilitated shotgun cloning of alpha-amylase and neutral protease genes from an unidentified Bacillus sp. in Bacillus subtilis by polyethylene glycol-induced protoplast transformation. This modification and the use of the plasmid vector pPL603b enabled us to simultaneously select for promoter-bearing recombinant plasmids that expressed amylase or protease activity. The inserts were found to be 4 and 4.6 kb, respectively. Although protease activity directed by the cloned gene was only 2- to 4-fold higher than for the donor strain, that of alpha-amylase was 28-fold higher.

Bacillus subtilis↗
PubMed1990

Protoplast fusion in microtiter plates for expression cloning in mammalian cells: demonstration of feasibility using membrane-bound alkaline phosphatase as a reporter enzyme.

Protoplast fusion is a method for directly transferring cloned DNA from bacteria to mammalian cells at high efficiency. Here, we have used membrane-bound alkaline phosphatase as a reporter enzyme in a miniprotoplast fusion assay. This work demonstrates the principle that large numbers of protoplast fusions can be done simultaneously and successfully, to assay for an activity encoded by an expression vector. The technique described here circumvents key hurdles to expression cloning. This method does not require a highly sensitive assay or a way of separating a rare expressing cell from the mixture of transfected cells containing other transfected genes. With a strong promoter, the protein encoded by the undiluted transfected cDNA should be produced at at least as high a level as it is endogenously produced in the cell from which its activity was first detected. Reference clones are stored, avoiding the need to separate out the cells that are successfully transfected; this also avoids the need to repurify the DNA from the transfected cell. Because of the use of microtiter plates, it is likely that such a method could be partially automated for many types of assays.

Alkaline Phosphatase↗
PubMed2004

Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells.

The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 degrees C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.

Cells, Cultured↗
PubMed2004

A Ca(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis: possible involvement in the C(4)-phosphoenolpyruvate carboxylase phosphorylation cascade.

In mesophyll cells (MC) of Digitaria sanguinalis, the C(4)-phosphoenolpyruvate carboxylase (C(4)-PEPC) initiating the photosynthetic pathway is controlled by a complex light-dependent phosphorylation process. We showed previously that the transduction cascade involves the phosphoinositide pathway and a Ca(2+)-dependent step, which precedes the upregulation of the PEPC kinase (PEPCk). We have now further characterized the cascade component requiring Ca(2+). A Ca(2+)-dependent protein kinase that shows several characteristics of the conventional type of mammalian protein kinase C (PKC) was detected in protein extracts from mesophyll cell protoplasts (MCPs). It catalyzed the in vitro phosphorylation of the C1-peptide PKC substrate and was markedly inhibited by a PKC-specific pseudosubstrate domain. However, it was only modestly activated by the phospholipids phosphatidylserine and lysophosphatidylcholine, while choline, oleyl acetylglycerol, phosphatidylinositol, and the phorbol ester phorbol 12-myristate 13-acetate did not show any effect. Nevertheless, its activity was found to be associated with a polypeptide of 75kDa that was recognized by a PKC antibody raised against the C-terminus of rabbit PKCbeta II. In addition, this protein kinase was also inhibited by the Ca(2+)-dependent protein kinase (CDPK)/PKC inhibitors W7, H7, and staurosporine. Surprisingly, it was found to be phosphorylated in dark-adapted MCPs, albeit to a low extent, and this did not change during protoplast induction by light. W7, H7, and staurosporine were shown to markedly inhibit C(4)-PEPC phosphorylation in light-treated MCPs. These results support the view that this protein kinase is a good candidate to represent the Ca(2+)-activated component of the C(4)-PEPC phosphorylation cascade.

Animals↗
PubMed2006

Transport of antimony salts by Arabidopsis thaliana protoplasts over-expressing the human multidrug resistance-associated protein 1 (MRP1/ABCC1).

ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of "safe-food" strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.

Antimony↗
PubMed2006

Genomic response programs of Candida albicans following protoplasting and regeneration.

Transcription profiling of Candida albicans cells responding to the elimination of the wall (protoplasts) and posterior regeneration was explored. DNA microarrays were used to measure changes in the expression of 6039 genes, and the upregulated genes during regeneration at 28 degrees C were assigned to fourteen categories. A total of 407 genes were upregulated during the process, of which 144 reached a maximum after 1 h. MKC1, a gene encoding a member of the regulatory pathway involved in cell wall integrity was overexpressed. Time-dependent expression divided the genes into 40 clusters. Clusters 1-19 were highly expressed initially (time 0) and downregulated following incubation, whereas transcription of the genes grouped into clusters 20-40 showed the opposite behaviour. These results suggest that the first clusters group genes permitting the cell adaptation to a sub-optimal environment due to removal of the wall, whereas the second group represents genes required for protoplasts regeneration after shifted to optimal conditions from 4 to 28 degrees C.

Candida albicans↗