Incorporation Of 14C-amino Acids Research Articles

The Use of an In Vitro-cultured Porcine Nasal Mucosa Model for the Biocompatibility Assessment of Biodegradable Magnesium

The development of an in vitro-cultured porcine nasal mucosa model is described. The model was subsequently used for the biocompatibility testing of resorbable magnesium-based implants, which are intended for use in the nasal cavity of patients with chronic rhinosinusitis (CRS). Test specimens made from either pure magnesium or titanium were incubated with the mucosal tissue for 48 hours. Afterwards, tissue viability, PGE2, IL-6 and IL-8 release, magnesium ion release, succinate dehydrogenase activity, apoptosis and 14C amino acid incorporation, were determined. The results suggested favourable biocompatibility, even in the case of rapidly-degrading pure magnesium. However, presumed effects on protein synthesis and apoptosis could not be confirmed.

Effects of salicylic acid on protein content and 14C-amino acid incorporation into proteins of pea roots

Pea (Pisum sativum L.) root treatment with salicylic acid (SA) changed the content of some proteins and incorporation of 14C-amino acids into proteins. The analysis of changes in these indices allowed us to subdivide all proteins into the four groups: (1) most abundant SA-independent proteins; (2) SA-dependent proteins, which content and 14C-amino acids incorporation both increased; (3) SA-dependent proteins, which content and 14C-amino acids incorporation both decreased; and (4) SA-dependent proteins, which content was not essentially changed (referred earlier to SA-independent proteins) but 14C-amino acids incorporation into these proteins was strongly suppressed. It is very likely that proteolysis of the proteins referred to the fourth group is very low and even a strong inhibition of their synthesis (incorporation of 14C-amino acids) does not result in the substantial decrease in their contents. Some SA-dependent proteins were identified by means of modern methods of proteomics: phosphoglyceromutase, S-adenosylmethionine synthase 3, enolase, chalcone isomerase, nucleoside diphosphate kinase 1, and tioredoxin h.

Metabolic signaling in aging and disease

The rate of biosynthesis and turnover of plasma protein fibrinogen is a marker of metabolic signaling in aging and disease. The rate in the young normal human subject of 0.260 mg/ml/24 hours increases to 0.378 in older normal subjects and to 0.466 in age matched coronary thrombosis patients measured by endogenous labeling of fibrinogen with L‐glutamic acid‐C14. This increased rate of fibrinogen turnover was traced to generation of occlusive fibrin polymers labeled by glycine‐C14 ethyl esters in the presence of active fibrin stabilizing factor. Circulating fibrin increased 520% (P<0.001) above normal in ischemic thrombotic cerebrovascular disease. Long chain saturated free fatty acids (FFA) exert not only primary control over incorporation of C14 amino acids into the fibrinogen structure but also activate the cascade sequence of reactions which convert fibrinogen into occlusive fibrin. FFA are normally transported by albumin to sites of energy metabolism. Decline in albumin synthesis in aging causes an increase in unbound FFA which induce not only increased fibrinogen synthesis but also activation of Hageman Factor generation of thrombin conversion of fibrinogen into occlusive fibrin. It has now been discovered that induction of a normal FFA/albumin ratio in plasma of coronary thrombosis and thrombotic stroke patients regenerates a normal level of fibrinogen synthesis by human hepatocytes.

Significant role for polysomes associated with the cytoskeleton in the control of protein synthesis during germination of triticale caryopses in the presence of abscisic acid

The influence of abscisic acid (ABA) on the process of polysome formation and synthesis of newly-formed proteins by different polysome populations was studied. Triticale caryopses were germinated in water or various ABA concentrations for 48 hrs, and afterwards they were transferred to a solution of 14C-amino acids and germinated for an additional 30 min. Embryos were separated from caryopses, and four polysome populations were isolated: the FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). ABA retarded both the process of polysome formation and their activity in forming new proteins in vivo in all studied fractions. Participation of polysomes in total ribosomal materials (sub-units, monosomes and polysomes) of each polysome population in the control sample was as follows: FP — 77; MBP — 72; CBP — 70 and CMBP — 66 %, whereas in sample treated by ABA (100 µM) it was accordingly: 17; 23; 27 and 28%. The largest population made up FP (in control sample 69%), participation of MBP was always lower and ranged from about 19 to 30 %. Participation of polysome populations bound with the cytoskeleton CBP and CMBP, both in control sample as well as in samples treated with 1 and 10 µM ABA solution, was only a few per cent. It should be noted that when the ABA concentration was higher (100 µM) (process of germination was strongly inhibited), participation of those two populations (CBP and CMBP) was much increased in embryos, respectively to about 18 and 20 %. In both the control group and in embryonal tissue treated with ABA increasing incorporation of radioactive precursors to newly-formed proteins in vivo in fractions of polysomes isolated by following buffers: C (FP), C + PTE (MBP), C + Tris (CBP) and buf. U (CMBP) was observed. It should be noted, that the biggest incorporation of 14C-amino acids into nascent polypeptide chains was found in the last polysome population (CMBP). In the sample treated with ABA (100 µM) the activity of this fraction (CMBP) in forming new proteins is several times, and in the case of FP dozens of times, more intense. Increased participation of CBP and CMBP in embryos of triticale caryopses treated with ABA (100 µM) and the largest incorporation of 14C-amino acids into nascent polypeptide chains synthesised by CMBP, may indicate the important role of proteins formed by polysomes associated with cytoskeleton in inhibition of germination and seedling growth by ABA.

Modifications of Intracellular Calcium Release Channels and Calcium Mobilization Following 70% Hepatectomy

The aim of this study was to investigate the properties of ryanodine and IP3receptors in regenerating liver following 70% hepatectomy, and to evaluate the hepatic Ca2+distribution and mobilization during this process. Specific [3H]ryanodine and [3H]IP3binding to hepatic smooth endoplasmic reticulum membranes, as well as subcellular Ca2+determination by atomic absorption flame photometry and Ca2+mobilization by INDO-1 AM spectrofluorescence in hepatocytes, was performed in regenerating livers after surgical 70% hepatectomy. Incorporation of14C amino acids into proteins and of32P into phospholipids was done in subcellular fractions. Ryanodine receptorKdpresented a dramatic increase after 12 h of surgery and remained high up to 2 days of treatment. IP3receptorBmaxshowed a significant augmentation starting at 6 h after hepatectomy and returning to normal values after 1 week. Cytosolic total calcium content decreased from 12 h until 4 days after hepatectomy whereas the microsomal and mitochondrial total calcium increased at 1 and 2–4 days of liver regeneration, which coincided with the differential turnover of proteins and phospholipids in these fractions. ATP-induced Ca2+transients in hepatocytes of 24-h-hepatectomized rats confirmed the altered sensitivity of the ryanodine receptor toward its ligand, since 10 times more ryanodine was necessary to alter the ATP-induced Ca2+transient. The data support the notion that the calcium release channels are targets of mechanisms of metabolic control during the proliferative response following 70% hepatectomy and might be part of the modified intracellular Ca2+dynamics during liver regeneration.

Possible involvement of the cytoskeleton in the regulation of barley caryopsis dormancy and germination

This study was conducted on barley cv. Ars. caryopses collected at full ripeness and divided into two batches. From one batch (dormant caryopses) polysomes were isolated from embryos immediately after harvesting and after two days of germination. From the other batch (non-dormant caryopses) the same was done after eight months storage in a dry state. A low ionic strength cytoskeleton-stabilizing buffer was used for the isolation of polysomes. Four different fractions of polysomes were examined: free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP). In germs grown from non-dormant caryopses, the first two fractions (FP + MBP) made up about 78 % of the total ribosomal material, whereas in embryos of dormant, imbibed caryopses, two last fractions (CBP + CMBP) made up about 71 %. The percentage of polysomes after 48 hours of imbibition of dormant caryopses in the FP, MBP and CBP was only about 13 % (i.e., 87 % monosomes), whereas a greater proportion (19.4 %) was found in the CMBP. The highest incorporation of 3H-uridine and 14C-amino acids (after 48 hours of germination and 0.5, 3 and 6 hrs incubation with precursors) took place in trhc CMBP both in dormant and non-dormant caryopses The major amount of the two polysome fractions associated with the cytoskeleton (CBP and CMBP) and the higher activity of CMBP in protein synthesis in embryos of dormant, imbibed triticale caryopses may indicate a significant role for polysomes associated with the cytoskeleton in the control of protein synthesis in dormant and germinating caryopses.

Fetal mouse salivary glands and applications for determining teratogenic mechanisms in vitro

Culture systems offer advantages for studying the actions of chemicals on mechanisms of development. Growth, [ 3H]thymidine incorporation, and 14C-amino acid incorporation were measured to determine culture conditions and the response of glands to three chemicals. Eagle's MEM supported the best growth and the best time for double isotope presence was the second 24 h of culture. Cyclamate had no effect on growth or isotope incorporation but cycloheximide disturbed 14C more than 3H. 5-Azacytidine was toxic but the incorporation of both isotopes exhibited the same pattern.

Repeated treatment with dithranol induces a tolerance reaction in keratinocytes in vitro

The hyperproliferative human keratinocyte line (HaCaT) was tested for dithranol tolerance (tachyphylaxis) at the cellular level. At day 4 after seeding, keratinocytes were treated with 0.3 or 1.0 microM dithranol. Data were compared with those from experiments including additional pretreatments with 0.3 microM at day 3, or at days 1 and 3. Protein content, DNA synthesis and protein synthesis (incorporation of 3H-thymidine and 14C-amino acids per protein) were determined at 24, 48 and 72 h after the last drug exposure. Protein content of attached cells decreased in relation to dose and frequency of treatments. Inhibition of DNA and protein synthesis (38.2% and 32.3%, respectively) also occurred 24 h after a single treatment with 0.3 microM dithranol, but was only 18.4% and 9.1% after pretreatment twice with 0.3 microM dithranol. This tolerance reaction in vitro, after repeated dithranol exposure of human keratinocytes, may be explained by a selective loss of drug-sensitive cells.

Lactate dehydrogenase release as an indicator of dithranol-induced membrane injury in cultured human keratinocytes

HaCaT cells, a rapidly multiplying human keratinocyte line, were tested for their sensitivity to antipsoriatic dithranol with regard to classical proliferation parameters and for the drug's action on the plasma membrane integrity by the dose- and time-dependent release of cytosolic lactate dehydrogenase (LDH). In the case of 3H thymidine as well as 14C amino acid incorporation the 50% inhibition concentration (IC50) was 0.2 microM dithranol 24 h after initial exposure to the drug. For protein content of attached cells the IC50 proved to be greater than 3.0 microM. Using 0.3, 1.0 and 3.0 microM dithranol, significant (p less than 0.05) dose dependent LDH release of 0.866 +/- 0.387, 1.842 +/- 1.127 and 2.938 +/- 1.635 mU per hour and cm2 confluent culture area was measured between the 5th and the 24th hour, compared to an acetone control of 0.504 +/- 0.299 mU/h x cm2. Between the 2nd and the 4th hour as well as from the 25th to the 48th hour and the 49th to the 72nd hour the LDH release after dithranol treatment did not exceed the control value. In accordance with these findings dose-dependent morphological signs of cell injury were detected by phase contrast microscopy beyond the 4th hour. The data reveal that: HaCaT cells are a very sensitive target for the antiproliferative action of dithranol; the drug causes considerable plasma membrane damage even at concentrations as low as 0.3 microM; and this membrane damage becomes evident after a latency of at least 4 h and for a limited period of up to 24 h.

A new model of epidermal differentiation: Induction by mechanical stimulation

In vivo, epidermal cells are committed to terminal differentiation in which they undergo a series of morphological and biochemical changes. In vitro, keratinocytes are able to undergo some steps of this differentiation process only. In view of the fact that in vivo skin is continuously subjected to mechanical stress, we investigated the stimulation of differentiation of transformed keratinocytes by mechanical stimulation. The cells, grown in plastic culture dishes, were periodically treated with weights exerting a pressure of 0.015 Ncm-2. This stimulation lasted from 1 to 4 days. Then keratinocytes were examined using indirect immunofluorescence, 3H-thymidine and 14C-amino acid incorporation, SDS polyacrylamide gel electrophoresis, and Western blotting. Following pressure treatment, the previously monolayered keratinocytes locally grew up to several layers, the number of horny scales increased and, after 4 days, the pattern of cytokeratin was modified. The total amount of keratin increased, forming granular accumulations, while the proliferation rate of the cells decreased. Both the 67 kDa and 49.5 kDa keratin subunits increased in stimulated cells. Moreover, a weak keratin band of 44 kDa appeared that was not present in controls. The results demonstrate that cyclic pressure promotes differentiation of cultivated epidermal cells.

Influence of blood sera from dogs subjected to ischemia and recirculation on incorporation of 14C-amino acids in vitro.

Incomplete ischemia of the spinal cord was produced in dogs by 40 min occlusion of the abdominal aorta that was followed by 5-40 min of recirculation. Amino acid incorporation into ribosomes in vitro in the presence of venous blood sera was estimated. The most significant reduction in incorporation was produced by sera of the dogs following a short recirculation period (5-10 min). No significant changes were observed at the end of the ischemic period nor at longer periods of recirculation. The decrease in incorporation might be the consequence of inactivation or absence of a substance stimulating polypeptide synthesis in vitro, normally present in blood sera of intact dogs, that temporarily loses its activity during recirculation.

In vitro synergism between hybrid immunotoxins and chemotherapeutic drugs: relevance to immunotherapy of prostate carcinoma.

Cultured prostate carcinoma cells incubated in the presence of a novel hybrid immunotoxin and ricin A chain exhibited synergy with the chemotherapeutic drugs vinblastine, methotrexate, and bleomycin. No cooperative effect was noted with adriamycin. Under conditions where individual components of immunotoxin or chemotherapeutic drug mixtures were nontoxic or minimally toxic the immunotoxin-drug mixture exhibited marked impact on 14C amino acid incorporation into prostate carcinoma cells. Analysis of drug-treated cells by flow cytometry indicated that cells exposed to vinblastine and bleomycin bound hybrid immunotoxin antibody to a greater extent than cells not exposed to these drugs. Adriamycin did not exhibit synergistic cytotoxicity with hybrid immunotoxin. Also, adriamycin did not enhance antibody binding as evaluated by flow cytometry. The fact that hybrid monoclonal antibody-ricin A chain (HIT-RAC) conjugates inhibited uptake of 14C amino acids 3 to 10-fold within 48 h of incubation with target cells and that this inhibition was further increased 2 to 3-fold in conjunction with three out of four chemotherapeutic drugs tested may be attributed to the unique cytotoxicity imposed by the hybrid immunotoxins. The RAC moiety is not chemically coupled to antibody but instead occupies one of the antigen-combining sites of the molecule. In this manner, RAC is closely juxtaposed to the cell membrane of the target cell and is anchored in this position via binding of the remaining antigen-combining site to p40 prostate restricted antigen.

Effects of methylglyoxal-bis (guanylhydrazone) and abscisic acid on polyamine metabolism in embryonectomized barley seeds

Incorporation of L-[U-14C] aiginine or L-[U-14C] ornithine into putrescine (Put), spermidine (Spd) and spermine (Spm) in embryonectomized barley seeds(Hordeum vulgare L. cv. Himalaya) was studied following imbibition with methylglyoxal-bis (guanylhydrazone)(MGBG) and abscisic acid (ABA). Both radiolabeled amino acids were incorporated into the amines as a result of: active polyamine biosynthesis in the seed during imbibition. In the aleurone layer, the Spd and Spn existed mainly in the free form (acid soluble). However about 50% of Put was recovered in conjugated form (s) (acid insoluble). Imbibition with 5 and 10μM ABA for 3 days increased the accumulation of the tree form of 14C-Put. probably as a result of inhibition (70%) of 14C-Spd accumulation. The ABA treatment showed no significant effect on levels of the conjugated form of Put and Spd. Imbibition with millimolar concentrations of MGBG resulted in (i) abnormal accumulation of the free form of Put and incorporation of 14C-amino acids into the diamine, (ii) progressive inhibition of the accumulation of the free forms of 14C-Spd and Spm. and (iii) reduction of the 14C incorporation into the conjugated forms of Put and Spd. Uptake of 14C-amino acids was not affected by MGBG treatment. The results indicate that MGBG may inhibit not only the synthesis of Spd and Spm, but the catabolism (e.g. oxidation) of Put in the aleurone layer.

Photo-induced toxicity of north sea crude oils toward bacterial activity

Toxicity of photolysis products from Ekofisk and Statfjord crude oils toward natural assemblages of marine bacteria was investigated. Incorporation of 14C-glucose, 14C-amino acids or 3H-thymidine were used as indicators of microbial activity. While none of these measurements revealed any significant effects of non-illuminated oil, a toxic effect was found after a few hours illumination of an oil slick.

Effect of chronic ethanol ingestion on liver and plasma protein synthesis in galactosamine-induced hepatitis in rats.

In rats a single injection (330 mg/kg) of D-galactosamine significantly inhibited in vivo incorporation of 14C-amino acids into fibrinogen, seromucoids and total liver proteins, but not albumin, by one third. Prior chronic exposure to ethanol (5 g/kg/day for 6 weeks) potentiated the inhibition of synthesis of all protein fractions (including albumin) up to about 50%. Propylthiouracil had no beneficial but deleterious effect against this potentiation of hepatotoxicity.

Bacteriostatic and bactericidal modes of action of bis(tributyltin)oxide on Legionella pneumophila.

The modes of action of bis(tributyltin)oxide (TBTO) doses between 1 X 10(4) and 6 X 10(7) molecules per cell on a single environmental isolate of Legionella pneumophila were studied by monitoring the following parameters: (i) growth, (ii) cell viability, (iii) 14C-amino acid incorporation, (iv) 14CO2 production from 14C-amino acids, (v) [3H]uridine incorporation, (vi) [3H]thymidine incorporation, (vii) oxygen consumption, (viii) cellular ATP levels, and (ix) adenylate energy charge. The amount of TBTO associated with the cells in these laboratory cultures was also compared with that remaining in the suspending medium. Most of the TBTO (68 to 88%) was found to be associated with the cells. This result explained why the cellular responses which were measured did not correlate with the TBTO concentration, but rather with the dose of TBTO to which the cells were exposed. At the lower TBTO doses tested (10(4) to 10(7) molecules per cell) a log-normal relationship was observed between the reduction in growth rate and the TBTO concentration. At intermediate TBTO doses (ca. 10(7) molecules per cell) growth stasis occurred, with nearly 100% of the cells in these cultures remaining viable for at least 5 h after treatment. The cellular function which seemed to be primarily affected at these levels of TBTO was the energy conversion mechanism, since the decline in the rates of CO2 production, oxygen consumption, and macromolecular synthesis was preceded by an immediate (within 1 min) drop in the intracellular levels of ATP and the adenylate energy charge. At the higher TBTO doses greater than 10(7) molecules per cell) an initial, precipitous, drop in the number of viable cells was observed, which was followed by a further exponential reduction of viable cells in the treated culture. This dramatic increase in bactericidal activity with a slight increase in the TBTO dose indicated that the modes of bacteriostatic and bactericidal action of TBTO were different.

Biosynthesis of branched-chain fatty acids from branched-chain amino acids in subcutaneous tissue of the marine little toothed whale, Stenella caeruleo-alba

1. 1. The conversion mechanism for 14CO 2 from [ 14C]Leu, [ 14C]Ile and [ 14C]Val and the incorporation of 14C-amino acids into lipids of subcutaneous tissue (melon and blubber), liver and muscle of the adult whale, Stenella caeruleo-alba were studied using a tissue culture method. 2. 2. Iso-acids such as -5:0, -11:0; -13:0 and -15:0 in melon triglycerides were biosynthesized from [ 14C]Leu. On the other hand, anteiso-acids such as -5:0, -11:0, -13:0, -15:0 and n-11:0 – n-16:0 acids were derived from [ 14C]Ile. 3. 3. Iso-acids such as -4:0, -12:0, -14:0 and 16:0 were biosynthesized from [ 14C]Val. 4. 4. Analytical data indicate that the compositions of these acids biosynthesized were similar to that of fatty acids in whale oil. 5. 5. The branched-chain fatty acid biosynthesized in blubber was iso-5:0 acid from [ 14C]Leu, with no long-chain branched acids, but n-12:0, -14:0 and -16:0 acids were also biosynthesized. 6. 6. Radioactivities derived from 14C-amino acids incorporated into lipids in liver and muscle were extremely low compared with those in subcutaneous tissue. 7. 7. The ratios of 14C-lipids/ 14CO 2 in every 14C-amino acid incorporation were higher in subcutaneous tissue than in liver and muscle, and especially in the case of [ 14C]Leu, the ratio in mclon was quite high. 8. 8. The incorporation of the 14C-amino acids into short- and long-chain acids and alcohols of wax esters was recognized in all experiments.

Nerve trophic effects: Partial purification from chick embryo brains of proteins that stimulate protein synthesis in cultured newt blastemata

Eighteen-day embryonic chick brain homogenates stimulate the incorporation of 14C-amino acids into proteins of cultured early to medium bud stage newt blastemata. DEAE-cellulose column chromatography of the homogenate yields a fraction which is highly active but unstable. Electrophoresis of the homogenates on “native” polyacrylamide gels (under nondenaturing conditions) yields two regions of activity on the gels when gel fractions are assayed by cutting the gels into segments and placing them directly in the culture medium. The relative mobilities ( R m ) of these active regions in 10% polyacrylamide gels are 0.45 ≤ R m ≤ 0.55 and 0.80 ≤ R m ≤ 0.90. On 7.5% gels the mobilities increase to 0.60 ≤ R m ≤ 0.75 and 0.95 ≤ R m ≤ 1.00. When brain homogenates are further fractionated by two-dimensional electrophoresis, the first dimension on “native” gels and the second dimension on gels containing SDS, only five visibly stained polypeptides can be found in the regions that correspond to the two regions of neurotrophic activity on the native gels. Four of these polypeptides with apparent molecular weights of 47,000, 36,000, 28,000, and 13,500 correspond to the slower migrating region on the native gels. In the faster migrating region, only one peptide of MW 13,500 is visible. We infer that the fast protein and one or more of the slow proteins stimulate protein synthesis in the newt blastemata assay and hence act as neurotrophic factors.

Distinctive characteristics of protein synthesis in maize embryos during the early stages of germination

Quiescent maize embryos were found to contain significant amounts of poly-A-rich pre-formed RNA. 14C-amino acid incorporation into trichloroacetic acid precipitable material was detected at slow rate at the begining of imbibition and fastly increased near 18 to 24 h. Polysomal formation was measured during this period. Addition of - amanitin to the incubation system at two 6h-pulse periods showed significant inhibition of the 14C-amino acid incorporation for the 18–24 h-period, but not for the 0–6 h-period.

Some Biochemical Effects of Glyphosate on Plant Meristems

Glyphosate promoted the activity of phenylalanine ammonia lyase (PAL) in both single node rhizome buds of Agropyron repens and root tips of wheat seedlings, in the latter case correlating inversely with decline in respiratory activity. Differential rates of 14C-amino acid incorporation suggest that glyphosate depleted the phenylalanine protein precursor pool with resultant inhibition of protein synthesis, as evidenced by a rapid decline in the level of 'soluble' protein. Promotion of PAL is assumed to occur by derepression through decline in end-product levels. Activities of shikimate:NADP oxidoreductase (SORase), chorismate mutase, and polyphenol oxidase (PPO) were also elevated, but to lesser extents than for PAL.