In vitro experiments concerning the state of polyphosphate in the yeast vacuole

1984 ◽  
Vol 30 (2) ◽  
pp. 236-246 ◽  
Author(s):  
J. J. Miller
Keyword(s):  
Amino Acids ◽  
Ribonucleic Acid ◽  
Dextran Sulfate ◽  
Insoluble Fraction ◽  
Yeast Cells ◽  
In Vitro Experiments ◽  
Yeast Vacuole ◽  
Low Concentrations ◽  
Cationic Amino Acids

Globules resembling the cytological entity volutin were produced in vitro by mixing solutions of polyphosphate and certain cationic substances known to occur abundantly in the yeast vacuole, i.e., S-adenosylmethionine, Mg2+, and K+. Other cationic substances found to form globules with polyphosphate were Mn2+, Zn2+, Ca2+, Cd2+, S-adenosylethionine, spermidine, and spermine. Cationic amino acids did not form a precipitate with polyphosphate and at low concentrations they inhibited precipitation of globules of polyphosphate–S-adenosylmethionine. Two other polyanions, dextran sulfate and ribonucleic acid, also formed globular precipitates with S-adenosylmethionine. Conditions affecting the formation and stability of polyphosphate–S-adenosylmethionine precipitates were studied in an attempt to reproduce certain characteristics of vacuolar polyphosphate "granules." Procedures commonly used for extraction of polyphosphate from yeast were applied to in vitro granules and the results indicated that part of the "acid-insoluble" fraction of the polyphosphate extracted from yeast cells may exist in combination with S-adenosylmethionine.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

scholarly journals Studies on Analogues of L-Cysteine and L-Cystine

Blood ◽  
1956 ◽  
Vol 11 (1) ◽  
pp. 1-10 ◽  
Author(s):  
AUSTIN S. WEISBERGER ◽  
LEIF G. SUHRLAND ◽  
JOSEPH SEIFTER
Keyword(s):  
Amino Acids ◽  
Spatial Configuration ◽  
Spatial Relationship ◽  
Inhibitory Effect ◽  
Selenium Compounds ◽  
Low Concentrations ◽  
Relationship Of ◽  
Normal Cellular Function

Abstract The amino acids L-cysteine and L-cystine appear to have an important role in the metabolism of leukocytes. Decreased availability of these amino acids may therefore have important effects on leukocytes. The possibility of decreasing the influx of radioactive L-cystine into leukemic leukocytes was investigated by exposing the leukocytes to various analogues of cysteine (cystine) prior to incubation with S35 L-cystine. It was found that a highly specific structural and spatial configuration is required to decrease the influx of S35 L-cystine. Thus unlabeled L-cysteine is effective in decreasing the incorporation of radioactive L-cystine. However, analogues of cystine in which there is modification or substitution of the sulfhydryl, amino or carboxyl group do not decrease the influx of S35 L-cystine. Furthermore, any alteration in the spatial relationship of the sulfhydryl and amino groups of L-cysteine also results in a loss of the ability of an analogue to decrease the incorporation of S35 L-cystine. Of the compounds studied and in the concentrations employed, only unlabeled L-cysteine, selenium cystine and phenyl selenium cysteine were effective. Selenium cystine is identical with cystine except that selenium replaces the sulfur in the molecule. Phenyl selenium cysteine is also closely related structurally to cysteine. The mechanism of action of selenium cystine and phenyl selenium cysteine in decreasing the influx of S35 L-cystine is not known. Other selenium compounds tested were ineffective. These compounds may exert their inhibitory effect by (a) competitive combination with specific intracellular receptors for L-cysteine (L-cystine), (b) inactivation of enzymes or compounds essential for normal cellular function, (c) alteration in membrane permeability or (d) a toxic effect of selenium. Since selenium cystine and phenyl selenium cystine are inhibitory in low concentrations in vitro, these compounds may have important effects on leukemic leukocytes in vivo.

scholarly journals Rates of aminoacyl-transfer-ribonucleic acid synthesis in vivo and in vitro by bean leaves

1970 ◽  
Vol 117 (5) ◽  
pp. 853-859 ◽  
Author(s):  
T. C. Hall ◽  
K. L. Tao
Keyword(s):  
Amino Acids ◽  
Ribonucleic Acid ◽  
Acid Synthesis ◽  
Leaf Discs ◽  
Endogenous Amino Acids ◽  
Intact Leaves ◽  
Aminoacyl Transfer ◽  
Bean Leaves

1. A procedure for measuring rates of aminoacyl-tRNA synthesis in vitro and in intact leaves is presented. 2. Leaf discs showed rates close to those of intact leaves. 3. Cell-free preparations showed similar rates when assayed by pyrophosphate exchange, but actual aminoacyl-tRNA formation rates appeared to be much lower. Evidence is presented that dilution of supplied labelled amino acids was a major factor causing the low apparent rates. 4. Attempts to strip endogenous amino acids from plant tRNA resulted in low acceptor capability of the tRNA.

De-nol for gastric and duodenal ulcers

1972 ◽  
Vol 10 (24) ◽  
pp. 95-96
Keyword(s):  
Amino Acids ◽  
Peptic Ulcer ◽  
Gastric Juice ◽  
Acid Medium ◽  
In Vitro Experiments ◽  
Duodenal Ulcers ◽  
Protein Mixture ◽  
Citrate Complex ◽  
New Treatment

De-Nol (Brocades) has been introduced as a new treatment for peptic ulcer. It contains tripotassium di-citratobismuthate. The makers claim that the bismuth is held within a citrate complex in a buffer. In the presence of amino acids and proteins, secondary chelates form, which are precipitated in an acid medium. It is claimed that the precipitate coats the surface of the ulcer crater with an insoluble chelate/protein mixture. In vitro experiments show that a heavy precipitate is formed if De-Nol is added to gastric juice, provided the pH remains below 5.0.

Na+-independent transport of bipolar and cationic amino acids across the luminal membrane of the small intestine

1997 ◽  
Vol 272 (4) ◽  
pp. R1060-R1068 ◽  
Author(s):  
B. G. Munck ◽  
L. K. Munck
Keyword(s):  
Amino Acids ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Short Circuit ◽  
Present System ◽  
Rabbit Ileum ◽  
Beta Alanine ◽  
Brown Adipose ◽  
Cationic Amino Acids

The role of sodium in transport of bipolar and cationic amino acids and their interactions were examined in vitro by measuring unidirectional influx across the brush-border membrane of intact rat jejunal and rabbit ileal epithelia. The chloride-dependent and beta-alanine inhibitable B(0,+) present in rabbit ileum was blocked by combining inhibition by beta-alanine with Na(+)- or Cl(-)-free conditions. Under these conditions, lysine influx across the brush-border membrane is Na+ independent. All Na+-independent influx of cationic and bipolar amino acids is by a system b(0,+) equivalent in the brush-border membrane of both species, where a system y+ is not present. System b(0,+) is shown to be a potent exchanger of intracellular leucine for extracellular lysine and of intracellular lysine for extracellular leucine. The model used to explain leucine stimulation of mucosa to serosa lysine transport can explain Na+ dependence of net lysine absorption. On the assumption that b(0,+) in situ, like the transporter induced by retroperitoneal brown adipose tissue in Xenopus laevi oocytes, acts as an obligatory exchanger, this model can also explain the effects of lysine on short-circuit current and net transport of sodium and the effect on transport capacity by preincubation at Na+-free conditions.

scholarly journals Amino acids attached to transfer ribonucleic acid in vivo

1975 ◽  
Vol 150 (3) ◽  
pp. 419-432 ◽  
Author(s):  
M Butler ◽  
A Darbre ◽  
H R Arnstein
Keyword(s):  
Amino Acids ◽  
Liquid Chromatography ◽  
Ribonucleic Acid ◽  
Rabbit Liver ◽  
Gas Liquid Chromatography ◽  
Tissue Protein ◽  
Flame Ionization ◽  
Total Tissue

1. tRNA was extracted from rabbit liver by both the phenol and diethyl pyrocarbonate methods under conditions preventing deacylation of the amino acids attached in vivo. 2. After deacylation 12 amino acids were determined by gas-liquid chromatography, by using the flame-ionization and nitrogen-sensitive thermionic detectors. 3. Comparison of the distribution of 12 amino acids attached to tRNA with those contained in total tissue protein and in the free pool showed little correlation. 4. Results for the enzymic charging assay for tRNA in vitro did not correlate satisfactorily with the analysis of amino acids attached to tRNA in vivo. Marked differences were ntoed in comparison made between our own and other published results.

scholarly journals The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

Effects of a strain of Saccharomyces cerevisiae (Levucell® SC), a microbial additive for ruminants, on lactate metabolism in vitro

1996 ◽  
Vol 42 (9) ◽  
pp. 927-933 ◽  
Author(s):  
Frédérique Chaucheyras ◽  
Gérard Fonty ◽  
Philippe Gouet ◽  
Gérard Bertin ◽  
Jean-Michel Salmon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Lactate Concentration ◽  
Lactate Production ◽  
Yeast Cells ◽  
Strictly Anaerobic ◽  
Streptococcus Bovis ◽  
Lactate Metabolism ◽  
Megasphaera Elsdenii

The effect of Levucell® SC, a strain of Saccharomyces cerevisiae marked as a feed additive for ruminants, was investigated in vitro on lactate metabolism by the ruminal bacteria Streptococcus bovis and Megasphaera elsdenii. The coculture between 107 live cells∙mL−1 of SC and a Streptococcus bovis strain in the presence of glucose reduced lactate production by the bacterial strain. Live yeast cells were able to compete with Streptococcus bovis for glucose utilization in strictly anaerobic conditions, so less glucose was available for the bacterium. SC also stimulated L-lactate utilization by a strain of M. elsdenii. The effect depended on the concentration of yeast cells added. Bacterial growth and fermentation end-product concentrations were also increased in the presence of SC. Some amino acids and vitamins, but not dicarboxylic acids, stimulated the bacterial specific activity of L-lactate uptake. SC was able to provide amino acids to M. elsdenii. In a coculture of Streptococcus bovis and M. elsdenii on glucose, the reduction of lactate concentration was improved by SC, the same trend being observed when maltose or soluble starch were used as carbon and energy source. These results indicate that SC can be a very useful tool to reduce lactate accumulation in vitro during fermentation of soluble sugars.Key words: rumen, Saccharomyces cerevisiae, lactate metabolism, Streptococcus bovis, Megasphaera elsdenii.

Influence of S-adenosylmethionine on DAPI-induced fluorescence of polyphosphate in the yeast vacuole

1980 ◽  
Vol 26 (8) ◽  
pp. 912-920 ◽  
Author(s):  
R. A. Allan ◽  
J. J. Miller
Keyword(s):  
Amino Acids ◽  
Brownian Motion ◽  
Aspartic Acid ◽  
Growth Medium ◽  
In Vitro Studies ◽  
Phosphate Solution ◽  
Yeast Vacuole ◽  
Glucose Phosphate ◽  
Ultraviolet Microscopy

Use of the fluorochrome 4′,6-diamidino-2-phenylindole∙2 HCl (DAPI) in ultraviolet microscopy revealed fluorescent objects in Brownian motion within the vacuoles of seven species of yeast. The abundance of these bodies increased when cells of Saccharomyees cerevisiae were transferred from growth medium to a glucose–phosphate solution, indicating that they contain polyphosphate. In addition, the effect on vacuolar fluorescence of supplementing a defined growth medium with amino acids provided evidence that they also contained S-adenosylmethionine. These deductions were supported by in vitro studies of the interaction and fluorescence of polyphosphate, S-adenosylmethionine, and DAPI. Vacuolar fluorescence of cells in suspension in glucose–phosphate solution was less after addition of exogenous arginine, lysine, or glutamine but not after addition of alanine, aspartic acid, or methionine.Mithramycin was superior to DAPI as a fluorochrome for ultraviolet demonstration of yeast nuclei since it stained the nuclei much more intensely and did not fluoresce with other material in the cells.

Development of a Resorbable Tricalcium Phosphate (TCP) Amine Antibiotic Composite

1987 ◽  
Vol 110 ◽  
Author(s):  
Lisa M. Morris ◽  
Praphyilla K. Bajpai
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Calcium Phosphate ◽  
Tricalcium Phosphate ◽  
Building Blocks ◽  
In Vitro Experiments ◽  
Dissolution Studies ◽  
Aluminum Calcium

AbstractCalcium phosphate collagen composites are currently being used to rebuild bone. Commercial collagen preparations have been claimed to he hvpo-antigenic but none are non-antigenic. The collagen in calcium phosphate collagen composites is eventually degraded to its component amino acids. Two thirds of collagen consists of glvcine, hvdroxvproline, and proline. Since both tricalcium phosphate (TCP) and collagen are resorbable and both can he utilized to form bone, incorporation of amino acids into TCP-amino acid composites should nrovide the building blocks for formation of new bone. Stich composites should also be non-antigenic. Previous work conducted with composites of amino acids with aluminum calcium phosphorous oxide (ALCAP) ceramics, has shown that these composites can be uised to repair traumatized bone in rats. In our study, experiments conducted with TCP cysteine, Ivsine, or proline composites showed that the composites, after setting to hardness, degrade slowly and can he molded into different froms. Dissolution studies were conducted with composites of TCP with each of the above amino acids in Tris-MCl buffer (PH 7.4) at 37°C in vitro. Experiments conducted with antibiotics showed that antibiotics such as penicillin and ervthromvcin can be incorporated into the TCP amino acid composite. Subcutaneous implants of TCP amino acid composites in rats showed that most of the TCP-proline composite was degraded bv day 4, TCP-lvsine composite he day 7, and TCP-cysteine composite by day 21. Our results suggest that TCP amino acid composites are biocomnatible and can he used to repair experimentally traumatized bone.

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