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1.
Norma L. Pucheu Norma L. Kerber Emilio A. Rivas Néstor Cortez Augusto F. Garcia 《Current microbiology》1997,34(3):155-161
Membranes from in vivo labeled cells of Rhodobacter
capsulatus U43[pTX35] grown photosynthetically carried 60% of
the [32P]-Pi in the “heavy” fraction (HM) after
sucrose gradient sedimentation. Metal-chelating chromatography of either
“heavy” or “light” (LM) membrane fractions rendered
similar Bchl-protein complex profiles after octyl-glucoside treatment,
including most of the radioactivity in the same corresponding elution
fraction (F II). Similar labeling distribution of pigment-protein complexes
was obtained for membranes of dark-grown cells induced by lowering oxygen
tension. Fractions derived from HM showed highly labeled LHIα, whereas the
same complex from LM was essentially [32P]-Pi-free, as revealed
by SDS-PAGE followed by autoradiography. Phospholipid analysis showed a
similar pattern for membranes isolated from cells photosynthetically or
semiaerobically grown, being the most abundant: phosphatidylglycerol,
phosphatidylethanolamine, cardiolipin, and phosphatidylcholine. Part of the
phospholipids from HM comigrated with LHIα during SDS-PAGE and dissociated
from the complexes only after solvent extraction and hydrophobic
chromatography. However, a small amount remained always attached to LHIα,
indicating an unusual strong interaction. These results suggest the existence
of two operationally defined membrane regions carrying LHIα complexes
differing in phosphorylation status and protein-phospholipid interaction.
Received: 10 August 1996 / Accepted: 10 September 1996 相似文献
2.
Zheng Jie Stephen J. Fey Henrik Hager Per Høllsberg Peter Ebbesen Peter Mose Larsen 《In vitro cellular & developmental biology. Plant》1990,26(10):937-943
Summary Primary cultures of trophoblasts established from human term placentae showed high viability and reproducibility. Two-dimensional
gel patterns obtained by metabolically labeling the trophoblasts with [35S]-methionine demonstrated that their pattern of gene expression was stable during the 6-d period investigated. Gel analysis
demonstrated the keratins 7, 8, 14, 17, 18, and 19. Analysis of the gel pattern confirmed the presence of a small proportion
of contaminating fibroblasts and lymphocytes. The gel patterns were compared with that of skin fibroblasts, peripheral lymphocytes,
and epithelial cells to identify a group of proteins that are enriched in the trophoblasts and thus may be used as marker
for these cells.
This work was supported by the Danish Cancer Society, the Lundbeck Foundation, the Danish Medical Research Council, “Pedersholmlegatet,”
and by “Anna og Jakob Jakobsens Legat.” 相似文献
3.
Mariangela Serra Enrico Sanna Alessandra Concas Cristina Foddi Giovanni Biggio 《Neurochemical research》1991,16(1):17-22
We report earlier that isoniazid and foot-shock stress individually increase the maximal number of [35S]TBPS binding sites (Bmax) measured ex vivo in unwashed membranes from rat cerebral cortex and that the increase due to both treatments are prevented by pretreatment in vivo with diazepam which alone induced a significant decrease in the total number of [35S]TBPS binding sites. In the present paper, the effect of stress was studied on both the increase in [35S]TBPS binding and the convulsant activity induced by isoniazid in unstressed rats. Isoniazid induced a time dependent increase in [35S]TBPS binding. The isoniazid-induced increase in [35S]TBPS binding was markedly potentiated by foot-shock stress. Moreover, foot-shock stress markedly reduced the latency to the appearance of generalized seizures induced by isoniazid (300 mg/kg s.c.). The results provide evidence that the in vivo inhibition of GABAergic transmission elicited by isoniazid results in an increase of [35S]TBPS binding in the rats cerebral cortex. The finding that stress, like isoniazid, enhances [35S]TBPS binding suggests that this treatment also inhibits the function of GABAergic synapses. 相似文献
4.
Extracellular polypeptides released by both N2-grown [peptide I] and NO3-grown [peptide II]Anabaena L-31 have molecular weight of approximately 3,500 but have distinctly different amino acid composition. Acid hydrolysis of
the peptide I fraction (obtained by separation on Sephadex G-25) yielded ten amino acids whereas that from peptide II fraction
yielded only 3 amino acids. On addition to a freshly inoculated N2-grown culture, the peptide I fraction stimulated pro-heterocyst and to a lesser extent heterocyst differentiation, whereas
the peptide II fraction strongly inhibited differentiation. The inhibitory effect of polypeptide II fraction could not be
relieved by methionine sulphoximine, which by itself enhances differentiation, but was greatly relieved by addition of the
peptide I fraction. The data suggest but does not prove, thatAnabaena L-31 synthesises “inducer” or “inhibitor” peptides which could possibly control pattern formation. 相似文献
5.
Junji Morimoto Shunsuke Imai Yuzo Taniguchi Yoshihiko Tsubura Howard L. Hosick 《In vitro cellular & developmental biology. Plant》1987,23(11):755-758
Summary A new murine mammary tumor cell line (BALB/c-MC) was established from a spontaneous mammary tumor in a 17-mo.-old female mouse
of the low mammary cancer strain BALB/cHe. The cell line was derived from a papillary adenocarcinoma. In monolayer culture
the line exhibits a pavementlike arrangement of cells and forms “domes” or “hemicysts” as the cells become confluent. The
cell line rapidly forms tumors when transplanted into young syngeneic BALB/cHe mice. The subcutaneous injection of 106 cells resulted in the development of mammary tumors (typical papillary adenocarcinomas) in 33 of 37 (87% recipients within
2 to 3 mo. after injection. These mammary tumors also metastasize to lung [14 of 33 (42%) of recipients] during this time.
The number of chromosomes in this cell line is hyperdiploid (average of 43, range 39 to 44). 相似文献
6.
Takayanagi T Hirokawa Y Yamamoto M Ohki T Fujiwara S Tsuzuki M 《Marine biotechnology (New York, N.Y.)》2007,9(1):56-65
Both coccolith-bearing cells (C-cells) and naked cells (N-cells) of the coccolithophorid Pleurochrysis haptonemofera can grow in salinities of more than 7‰ (about 20% of a “normal” sea water salinity [35‰]), with the highest growth rates
in salinities of more than 14‰. Microscopic observations of cells suspended in 100 mM NaCl (7‰) showed that, while N-cells
were swelling uniformly all over the cell surface, C-cells were bulging the plasma membrane from the hole of the coccosphere
at the apical (flagellar) pole of the cell. Effects of several cations and anions on the morphological change of C-cells under
hypoosmotic pressure were investigated. When 100 mM K+ was used, protoplasts were released from the coccosphere completely in almost all the cells. This phenomenon was shown with
K+ most effectively. The protoplasts could grow in the fresh medium and form the first coccolith within 9 h. 相似文献
7.
Mixed glial–neuronal cultures prepared from rat embryonic cortical cells were either treated with aracytosine or infected with an adenovirus encoding the Lac-Z gene according to two protocols of infection. In each experiment, 24 h before the end of the incubation period, [35S]methionine was added to one set of cultures which were performed in plastic chamber slides. At 10–13 days in vitro, control and treated cultures were processed either for immunocytochemical detection of neuron-specific enolase (NSE)-stained cells or for measurement of [35S]methionine incorporation. For the latter, cultures grown in the chamber slides were fixed with 4% paraformaldehyde, dehydrated, and air-dried. After removal of the upper structures of the chambers, the slides were directly transferred to a 1200 -imager, a gaseous detector which displays a digital image of the cultured cells and permits the quantitative measurement of incorporated [35S]methionine within a few hours. In aracytosine-treated cultures, we observed that the numbers of NSE(+) cells as well as [35S]methionine incorporation were decreased compared with control cultures. After viral infection, the number of NSE(+) neurons and the amount of radioactivity incorporated were either the same in control and infected cultures or decreased for the cultures treated according to the different protocols. In all cases, the amount of [35S]methionine incorporated varied in the same direction as the number of NSE(+) neurons in cultures. The digital imaging of the cultures permitted observation of the layer of cultured cells. It appears that such a rapid and direct measurement of incorporation of a radiolabeled indicator of protein synthesis may be considered as a quick and reliable marker of cell survival and/or proliferation. 相似文献
8.
The occurrence of the polarized synthesis of DNA in embryogenic cell clusters of carrot on the third and fourth days after
transfer to an embryogenesis-inducing medium was observed by labeling with [3H]thymidine and autoradiography. The cells that were actively synthesizing DNA were separated from cells that were not synthesizing
DNA by maceration of cell clusters into individual protoplasts and centrifugation in a Percoll density gradient. [35S]Methionine-labeled proteins extracted from the two types of cell were analyzed by SDS-PAGE and fluorography. Three polypeptides
(of 69, 98 and 108 kD, respectively) were found only in cells that were actively synthesizing DNA and could be candidates
for markers of the polarity of DNA synthesis that is specific to embryogenesis. 相似文献
9.
[(4-methoxy-4(3-β-d-galactose-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.02,7]tridec-2,7-ene] (“sβ-Gal 102”) and sodium [4-methoxy-4(3-β-d-glucuronic acid-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.02,7]tridec-2,7-ene] (“sβ-Glucor 102”) are carbohydrate-containing 1,2-dioxetane compounds that produce chemiluminescence upon
enzymatic hydrolysis by β-d-galactosidase, and β-d-glucuronidase, respectively. In this study, we have characterized and validated a sensitive detection principle for viable
Escherichia coli based on enzymatic cleavage of sβ-Gal 102 and sβ-Glucor 102 (“ColiLight II”). The proposed chemiluminescent assay was optimized
with respect to analytical requirements including incubation time, temperature, pH, enzyme induction, and cell permeabilization.
The sensitivity and specificity rates of the assay were tested on ten different bacterial genera. The assay was found to be
representative based on low coefficients of variations for both accuracy and precision. The analysis time was less than 1 h
and the analytical detection limit was 102 to 103
E. coli cells. In combination with membrane filtration and a brief resuscitation step of 4 h, the proposed assay was capable of detecting
low concentrations of stressed E. coli in potable water (<30 CFU 100 ml−1). The proposed chemiluminescent enzyme assay may be used for assessing the metabolic activity of E. coli in oligotrophic environments and for early warning detection of low concentrations of E. coli in water for human consumption. 相似文献
10.
Mengovirus RNA was translated in Ehrlich ascites cell extracts using as radioactive precursors f[35S]Met tRNAFMet and [35S]Met tRNAMMet to label the products in N-terminal and internal positions, respectively. Tryptic peptides were compared with those derived from purified [35S]Met-labeled mengovirus. The results indicate that the sequences corresponding to the viral coat polypeptides are preceded by a short “lead-in” peptide which is probably removed by a cleavage process in infected cells. 相似文献
11.
Summary. Hepatocyte preparations highly enriched in cells from either the periportal or the perivenous zone of the liver acinus were
prepared using a digitonin/collagenase perfusion method. Five enzymes of cysteine metabolism were assayed in both periportal
and perivenous preparations. The ratios of periportal to perivenous activity were 0.76, 0.60, 0.81, 1.62, and 1.01 for cysteine
dioxygenase, cysteinesulfinate decarboxylase, γ-glutamylcysteine synthetase, cystathionase, and asparate (cysteinesulfinate) aminotransferase, respectively. Only cysteinesulfinate
decarboxylase activity was significantly different between periportal and perivenous cells. In incubations with 2 mmol/L [35S]cysteine, total cysteine catabolism ([35S]taurine plus [35S]sulfate) between periportal and perivenous cells was not different, which is consistent with the observation of similar
cysteine dioxygenase activity across the hepatic acinus. Consistent with the lower cysteinesulfinate decarboxylase activity
in periportal cells, 16% of the total catabolism of [35S]cysteine in periportal cells resulted in taurine synthesis compared to 28% in perivenous cells. A lower rate of [35S]glutathione synthesis was observed in periportal cells compared to perivenous cells, but γ-glutamylcysteine synthetase activity was not significantly different between perivenous and periportal cells. Cysteinesulfnate
decarboxylase can be added to the list of enzymes whose activities are markedly enriched in perivenous cells.
Received January 15, 2002 Accepted February 4, 2002 Published online September 4, 2002
Acknowledgements This work was supported by the National Research Initiative Competitive Grants Program/United States Department of Agriculture
Competitive Research Grant 02-37200-7583.
Authors' address: Dr. Martha H. Stipanuk, Division of Nutritional Sciences, 227 Savage Hall, Cornell University, Ithaca, NY 14853-6301, U.S.A.,
E-mail: mhs6@cornell.edu 相似文献
12.
The metabolic pathway of primary carbon fixation was studied in a peculiar pennate marine diatom, Haslea ostrearia (Bory) Simonsen, which synthesizes and accumulates a blue pigment known as “marennine”. Cells were cultured in a semi-continuous
mode under saturating [350 μmol(photon) m−2 s−1] or non-saturating [25 μmol(photon) m−2 s−1] irradiance producing “blue” (BC) and “green” (GC) cells, characterized by high and low marennine accumulation, respectively.
Growth, pigment contents (chlorophyll a and marennine), 14C accumulation in the metabolites, and the carbonic anhydrase (CA) activity of the cells were determined during the exponential
growth phase. Growth rate and marennine content were closely linked to irradiance during growth: higher irradiance increased
both growth rate and marennine content. On the other hand, the Chl a concentration was lower under saturating irradiance. The distribution between the Calvin-Benson (C3) and β-carboxylation (C4) pathways was very different depending on the irradiance during growth. Metabolites of the C3 cycle contained about 70 % of the total fixed radioactivity after 60 s of incorporation into cells cultured under the non-saturating
irradiance (GC), but only 47 % under saturating irradiance (BC). At the same time, carbon fixation by β-carboxylation was
24 % in GC versus about 41 % in BC, becoming equal to that in the C3 fixation pathway in the latter. Internal CA activity remained constant, but the periplasmic CA activity was higher under
low than high irradiance. 相似文献
13.
Joelle Vilamitjana-Amedee Reine Bareille Francois Rouais Arnold I. Caplan Marie-Francoise Harmand 《In vitro cellular & developmental biology. Animal》1993,29(9):699-707
Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated
by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis
of [3H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin.
They respond to 1,25 dihydroxy vitamin D3 by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the
third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline
phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding”
structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was
associated with45Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance. 相似文献
14.
L. Ouelhazi M. Filali J. Creche J. C. Chénieux M. Rideau 《Plant Growth Regulation》1993,13(3):287-295
Total and neosynthesized proteins of periwinkle cell suspensions (Catharanthus roseus) were first investigated in cells grown in a 2,4-D-containing medium. Analysis of total (silver-stained) proteins by two-dimensional gel electrophoresis revealed that the levels of seventeen polypeptides were altered during the growth cycle of the cells. Analysis of in vivo [35S]-methionine labeled polypeptides revealed differences in the synthesis of at least 35 polypeptides. Three polypeptides with molecular masses of 30, 35 and 39 kDa appeared to be specific markers of the early stationary phase. In a second sequence of experiments, cells were grown in a 2,4-D-free medium. Alterations in protein synthesis were observed: several polypeptides were expressed earlier in the 2,4-D-starved cells than in control cells; the synthesis of at least two specific polypeptides was increased in cells grown in 2,4-D-free medium, whereas the synthesis of three other polypeptides (molecular masses 33, 34 and 52.5 kDa) was switched on in these cells. As previous studies showed that 2,4-D depletion increased the alkaloid production in C. roseus cells, the present results may suggest that these polypeptides are implicated in the regulation of the alkaloid pathway. 相似文献
15.
Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[3H]glucosamine, [35S]sulfate, and L-[14C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[3H]glucosamine, but not of [35S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[14C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect
on D-[3H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[3H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc
analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[3H]glucosamine and [35S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis,
namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at
1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures
(∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the
incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic
inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8
suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein
synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[3H]glucosamine and [35S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting
total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another
metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented. 相似文献
16.
Temperature up to 16‡C reduced endocytosis of [35S]-proteoglycans by human skin fibroblasts to less than 15% of that at 37‡C. At temperatures between 20–26‡C endocytosis was
more than 50%. At temperatures below 26‡C, the relative rate of degradation of endocytosed [35S]-proteoglycans was several fold less than the rate of endocytosis.
Codistribution of endocytosed [35S]-proteoglycans and the lysosomal marker enzyme Β-hexosaminidase upon subcellular fractionation indicated that endocytotic
vesicles containing [35S]-proteoglycans had fused with lysosomes at 37‡C and at 16‡C. The prolonged halflives of endocytosed [35S]-proteoglycans at 16–26‡C could not be explained merely by a temperature dependent reduction of catalytic activity of lysosomal
enzymes participating in the degradation of sulphated proteoglycans. 相似文献
17.
Functional activation of α2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [35S]-guanosine 5′-O-(γ-thiotriphosphate) ([35S]GTPγS) binding assay. α2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [35S]GTPγS binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However,
even after careful optimization of experimental conditions the specificity of ligands for rat α2 adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems
before achieving their maximal effect in the α2A-adrenergic system. Only using compromising concentration of agonist (up to 1 μM UK14304) and antagonist (up to 1 μM RS79948)
to inhibit agonist’s effect, allowed us to filtrate out α2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies
of this system for different animals from behavioral experiments. 相似文献
18.
Summary Wing imaginal discs from 7th instarGalleria mellonella L. larvae evaginate and exhibit tracheolar elongation when exposed to 20-hydroxyecdysone in vitro. This response was elicited
within 24 h of treatment as was a greater than fourfold stimulation of the incorporation of [3H]leucine into disc proteins. Autoradiographic analyses of [35S]methionine labeled polypeptides separated on two-dimensional gels, however, revealed no differences in protein profiles
between control and treated discs until 48 h following exposure to molting hormone. At this time, wing imaginal discs exposed
to 1 μg/ml 20-hydroxyecdysone synthesized four unique polypeptides not detected either in controls or in discs treated for
24 h. These four new proteins were also found to be synthesized by imaginal discs that had evaginated in vivo. These results
suggest that these proteins are normally synthesized subsequent to evagination and do not play a role in the morphological
events necessary for evagination.
Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA.
S. G. M. is employed through a cooperative agreement between the Insect Attractants, Behavior and Basic Biology Laboratory
and the Department of Entomology, University of Florida. 相似文献
19.
Baumler DJ Hung KF Jeong KC Kaspar CW 《Extremophiles : life under extreme conditions》2007,11(6):841-851
Acidophiles are typically isolated from sulfate-rich ecological niches yet the role of sulfur metabolism in their growth and
survival is poorly defined. Studies of heterotrophically grown “Ferroplasma acidarmanus” showed that its growth requires a minimum of 100 mM of a sulfate-containing salt. Headspace gas analyses by GC/MS determined
that the volatile sulfur compound emitted by active “F. acidarmanus” cultures is methanethiol. In “F. acidarmanus” cultures grown either heterotrophically or chemolithotrophically, methanethiol was produced constitutively. Radiotracer
studies with 35S-labeled methionine, cysteine, and sulfate showed that all three were used in methanethiol production. Additionally, 3H-labeled methionine was incorporated into methanethiol and was probably used as a methyl-group donor. Methanethiol production
in whole cell lysates supplied with SO32− indicated that NADPH-dependant sulfite reductase and methyltransferase activities were present. Cell lysates also contained
enzymatic activity for methionine-γ-lyase that cleaved the side chain of either methionine to form methanethiol or cysteine
to produce H2S. Since methanethiol was detected from the degradation of cysteine, it is likely that sulfide was methylated by a thiol methyltransferase.
Collectively, these data demonstrate that “F. acidarmanus” produces methanethiol through the metabolism of methionine, cysteine, or sulfate. This is the first report of a methanethiol-producing
acidophile, thus identifying a new contributor to the global sulfur cycle. 相似文献
20.
Yan Xu Haihui Ye Jun Ma Huiyang Huang Guizhong Wang 《In vitro cellular & developmental biology. Animal》2010,46(8):708-717
Crustacean neurons, obtained from the cerebral ganglion of the mud crab Scylla paramamosain, were successfully cultured in vitro. They maintained typical morphological characteristics and showed better outgrowth in
modified Medium 199 (M199) medium than that in Liebowitz’s L-15 medium. Fetal bovine serum (FBS), muscle extracts, and hemolymph
of the mud crab S. paramamosain were added as supplements. Only 20% FBS could promote neuron outgrowth, while muscle extracts and hemolymph of S. paramamosain did not improve neuron outgrowth. For cell dissociation, both collagenase type I and trypsin worked well as determined by
initial cell viability and following cell outgrowth potential. More than six kinds of cells with different morphological characteristics
were identified in the neuron outgrowth. They were “small cells”, “veilers”, “branchers”, “multipolar cells”, “super-large
cell”, and “bipolar cells”. Among all of the cells, bipolar cells were identified for the first time in crustacean neurons
culture and they could live longer than other cells. The neurons could grow for more than a week before retraction and eventual
degradation. 相似文献