Incorporation Of Radioactive Precursors Research Articles

Antitrypanosomal Action of Cis-Diamminedichloroplatinum (II) Analogs

The present report deals with the alterations produced by cis-diamminedichloroplatinum (II) (DDP), and 2 of its analogs: cis-Pt(II)(tranylcypromine)2Cl2 and cis-Pt(II)(benzothiazole)2Cl2 in cultured epimastigote forms of Trypanosoma cruzi. Studies have been performed at the ultrastructural level and the inhibitory effect of these complexes on macromolecule synthesis, evaluated by 3H-thymidine, 3H-uridine, and 3H-leucine incorporation, has been investigated. DDP at concentrations of 50 and 100 micrograms/ml does not inhibit significantly the incorporation of radioactive precursors, but a clear decrease was observed with the 2 analogs. Eight hours of treatment at a concentration of 10 micrograms/ml rendered in all 3 cases an increase in autophagic vacuoles and lipids as well as an abnormal condensation of the nucleus chromatin.

Increased axonal transport in peripheral nerves of thiamine-deficient rats

Thiamine deficiency has been implicated as a signifccant contributing factor in the development of peripheral neuropathies in chronic alcoholic patients. We hypothezized that thiamine deficiency may lead to an alteration in axonal transport because it has been associated with “dying-back” neuropathies and its importance in neural tissue has been demonstrated with antimetabolites. To test this possibility rats were made thiamine-deficient by feeding a liquid diet lacking thiamine. Control rats were pair-fed a complete liquid diet. The deficiency developed after 3 to 4 weeks and was evidenced by anorexia, weight-loss, and a significant increase in the erythrocyte transketolase activity ratio. Also, the sural nerve conduction velocity was found to be significantly reduced in these animals (18.74 m/s) relative to that of pair-fed control rats (31.99 m/s). In vitro transport experiments utilizing dorsal root ganglia-sciatic nerve preparations indicated that twice as much [ 35S]methionine-labeled protein accumulated at a ligation by fast transport in the thiamine-deficient rats as in nerves of their pair-fed controls. There was no difference in the level of incorporation of radioactive precursor into the dorsal root ganglia. The increase in transport suggests that thiamine deficiency per se has no detrimental effects on the transport machinery and process, but may indicate extensive regenerative activity in the distal portions of these axons.

The role of sera, growth factors, and hormones in the in vitro production of disaturated phosphatidylcholine and propagation of undifferentiated type II alveolar cells from the fetal rabbit lung.

Undifferentiated type II alveolar cells isolated from the fetal rabbit lung were cultured in chemically-defined medium supplemented with 10% carbon-stripped fetal bovine serum for 2-3 days and then placed in serum-free medium, or medium supplemented with either 10% carbon-stripped fetal bovine serum (sFBS) or 10% unstripped fetal bovine serum. Cells exposed to the latter treatment showed the highest growth rate but the lowest level of [3H]choline incorporation into disaturated phosphatidylcholine (DSPC). Cells in serum-free medium showed the lowest rate of growth but removal of serum components stimulated a rapid increase in radioactive precursor incorporation. Cells exposed to the stripped serum were intermediate in both measurements. Incubation of undifferentiated type II cells with EGF stimulated growth but only in the presence of sFBS. EGF also stimulated [3H]choline into DSPC after 48 hours of exposure to the peptide. This latter response could only be elicited in serum-free medium. In the presence of sFBS no effect of the peptide was detected on radioactive precursor incorporation. Similarly dexamethasone (0.55 nM) or 20% medium which had been conditioned by fetal lung fibroblast monolayers did not stimulate [3H]choline in DSPC in the presence of sFBS. In contrast these agents did evoke a significant increase in radioactive precursor incorporation when the undifferentiated type II alveolar cells were incubated in serum-free medium.

“Matrigenin” activity from bovine bone—III. Effects on glycosaminoglycans and proteoglycans of human synovial cells in culture

1. 1. Human synovial fibroblastic cells were cultured in the presence and absence of an extract from bovine bone containing “matrigenin” activity. The rate of incorporation of radioactivity into the glycosaminoglycans of the medium of “matrigenin”-treated cultures increased after 24 hr of incubation, compared to “controls”. 2. 2. Higher serum concentrations had a greater effect on the incorporation of radioactivity into hyaluronic acid synthesized by “matrigenin”-treated cultures, than by “controls”. 3. 3. Incorporation of radioactive precursors into the proteoglycans isolated from the medium was greater in the “matrigenin”-treated cultures than in “controls”. The synthesis of a large mol. wt proteoglycan was specifically stimulated.

Biosynthesis of glycosaminoglycans by trabecular meshwork cells in vitro.

We compared the incorporation of radioactive precursors into glycosaminoglycans by cynomolgus monkey trabecular meshwork cells in tissue culture and in organ culture. Hyaluronic acid and a variety of sulfated glycosaminoglycans were synthesized in both systems. In organ cultures, the ratio of chondroitin 6- to chondroitin 4-sulfate was higher than that found in tissue cultures. Also, a greater proportion of total glycosaminoglycans found in the organ culture medium was represented by hyaluronic acid. The higher production of chondroitin 6-sulfate and hyaluronic acid, as noted in some embryonic systems, suggested that cells in organ cultures may resemble an acute wound healing or an early developmental state more closely than did cells in tissue cultures.

Mechanism of antifungal action of citrinin.

The mycotoxin citrinin had antifungal activity under acidic conditions. At the minimum inhibitory concentration, it completely inhibited cellular respiration and partially inhibited the incorporation of radioactive precursors into macromolecules in Saccharomyces cerevisiae. It had no effect on cell permeability. In mitochondrial preparations, it significantly inhibited succinate oxidase and NADH oxidase. Rhizopus chinensis was more sensitive than S. cerevisiae; its growth and mycelial respiration at acidic pH were completely inhibited by lower concentrations of citrinin. The pH-dependent antifungal activity of citrinin seems to be associated with its uptake by fungi. Approximately half of the citrinin taken up was found in mitochondria. The main site of the antifungal action of citrinin, therefore, appears to be the mitochondrial electron transport system.

Response of Transplantable Tumors in Mice and of Macromolecular Synthesis to 17β-Acetamido-3-aza-A-homo-4α-androsten-4-one

17 beta-acetamido-3-aza-homo-4 alpha-androsten-4-one has cytostatic activity against Ehrlich ascites tumor, L1210, and P388 leukemias in mice when administered intraperitoneally. The effect of the homo-aza-steroid on the incorporation of radioactive precursors to DNA and RNA of L1210 leukemia cells was investigated. It was found that treatment of cells with 12.5 micrograms/ml of the drug for 1 hour inhibited DNA synthesis by 81%. This was partly because the drug affected the radioactive thymidine pool in the cell. The inhibitory effect was found to be reversable. The incorporation of [3H]thymidine into DNA was lower when cells were incubated in the presence of S9 mix. It was also found that the same compound inhibited RNA synthesis by 67%.

Response of Transplantable Tumors in Mice and of Macromolecular Synthesis to 17β-Acetamido-3-aza-A-homo-4α-androsten-4-one

17β-acetamido-3-aza-A-homo-4α-androsten-4-one has cytostatic activity against Ehrlich ascites tumor, 11210, and P388 leukemias in mice when administered in-traperitoneally. The effect of the homo-aza-steroid on the incorporation of radioactive precursors to DNA and RNA of 12210 leukemia cells was investigated. It was found that treatment of cells with 12.5 fig/ml of the drug for I hour inhibited DNA synthesis by 81%. This was partly because the drug affected the radioactive thymidine pool in the cell. The inhibitory effect was found to be reversable. The incorporation of [3H]thymidine into DNA was lower when cells were incubated in the presence of S9 mix. It was also found that the same compound inhibited RNA synthesis by 67%.

Propagation of infectious bovine rhinotracheitis (bovine herpes-1) virus in murine primary cell cultures

Virus synthesis in BALB/C mouse lung and kidney primary cultures infected with infectious bovine rhinotracheitis (IBR) virus started between 6 and 8 hours after virus inoculation and reached a maximum titer of 5.5 log10 plaque forming units (PFU) at 48 hours postinfection (PI). Cytopathic effect (CPE) in cell cultures occurred at 8-10 hours and over 90% of the cells had CPE by 48 to 72 hours PI. The bulk of the newly replicated virus (60-80%) was cell-associated as determined by plaque assay of extracellular and intracellular virus. Pulse-chase experiments demonstrated incorporation of radioactive precursors into viral DNA and protein macromolecules. Viral DNA synthesis was initiated between 2 and 4 hours PI and was maximum at 4 to 6 hours. Viral proteins were detected at 4 hours and peaked between 6 and 8 hours PI. Enzyme-linked immunosorbant assay (ELISA) confirmed synthesis of specific viral proteins, which gradually increased during the virus growth cycle. Abstract in French is given at the end of the article.

Increased synthesis of membrane macromolecules is an early response of retinal neurons to trimethyltin intoxication

We studied the synthesis and axonal transport of proteins and glycoproteins in the visual system of adult Long-Evans rats that had received 4 weekly doses of trimethyltin hydroxide (TMT, 4 mg/kg b. wt.) by gastric intubation. One week following the last dose, an in vitro assay was used to study the rate of incorporation of radioactive precursors into various macromolecules of isolated retinas. Retinas from TMT-treated rats showed increased apparent rates of synthesis, relative to retinas from control rats, for proteins [( 35S]methionine precursor) and glycoproteins [( 3H]fucose precursor). Gel electrophoretic analysis of newly synthesized proteins indicated that the increased synthesis was a generalized effect, i.e. it was not restricted to a select subset of proteins. The axonal transport of these macromolecules by retinal ganglion cells to axons (optic tract) and nerve endings (superior colliculus) was examined in vivo following intraocular precursor injection. The amount of material transported, relative to that synthesized in the retina, was not appreciably altered in TMT-treated rats, indicating that TMT did not selectively impair axonal transport. The biochemical changes were accompanied by minimal ultrastructural alterations and little neuronal necrosis in the retina. We suggest that TMT induces increased synthesis of membrane macromolecules in retinal neurons; this may reflect an early reactive (compensatory) response rather than a regressive (degenerative) response of retinal neurons to TMT. Our data do not support the hypothesis that TMT induces a functional impairment of neuronal endoplasmic reticulum or Golgi apparatus.

Biosynthesis and Turnover of Cell Wall Glycoproteins during the Vegetative Cell Cycle of Chlamydomonas reinhardii

Abstract Biosynthesis and turnover of the different cell wall components have been studied during the vegetative cell cycle of Chlamydomonas reinhardii by pulse-labelling with [3H]proline and [35S]methionine and by pulse-chase experiments. Two phases of biosynthesis of insoluble cell wall material could be distinguished: 1. de novo synthesis of the daughter cell walls during cytokinesis and 2. cell wall enlargement during cell growth. During the cell enlargement period, a turnover of the insoluble wall component was observed. The released fragments were found to be accumulated in the culture medium. The LiCl-soluble cell wall glycoproteins were found to be precursors of the insoluble cell wall layer. Biosynthesis of the LiCl-soluble cell wall glycoproteins was observed mainly during the time period between cytokinesis and the end of the following cell enlargement period. Labelling of all the cell wall components was found to be strongly reduced during the time period between the end of the growth phase and cytokinesis. During cytokinesis, labelling of the insoluble cell wall material preceded the incorporation of radioactive precursors into the LiCl-soluble wall fraction.

Differentiation-related changes in glycoprotein synthesis by human keratinocytes.

Human keratinocytes were cultured in media in which the Ca2+ concentration controlled the stage of differentiation. In media containing less than 0.1 mM-Ca2+ keratinocytes grew as a monolayer, but in the presence of 2mM-Ca2+ the cells differentiated and formed stratified colonies. Glycoproteins of both stratified and unstratified cells were radiolabelled by metabolic incorporation of radioactive precursors and by cell-surface labelling using galactose oxidase/NaB3H4. The radiolabelled keratinocytes were extracted with 0.5% Triton X-100, and the glycoproteins in both the Triton X-100-soluble and Triton X-100-insoluble fractions were analysed by polyacrylamide-gel electrophoresis in the presence of SDS. Two Triton X-100-soluble glycoproteins with high Mr values (greater than 200,000) were major glycoproteins in stratified keratinocytes, but were present in only trace amounts in unstratified keratinocytes. Characterization of these glycoproteins by examination of the effect of tunicamycin on their synthesis and the effect of neuraminidase on their migration characteristics showed that they were cell-surface sialoglycoproteins containing O-glycosidically linked oligosaccharides. Analysis of the adherent cytoskeletons left after Triton X-100 extraction of stratified and unstratified keratinocytes revealed that a glycoprotein of Mr 184,000 was decreased in stratified keratinocytes. Incubation of unstratified keratinocytes in high-Ca2+ medium resulted in a rapid modification of the glycoprotein of Mr 184,000, and it is suggested that this event may be related to desmosome formation and stratification.

Gonadotropin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacyglycerol in rat granulosa cells: further evidence for the involvement of Ca2+ and protein kinase C in the action of GnRH.

GnRH provokes a phospholipid response in rat granulosa cells that has been characterized by increased incorporation of radioactive precursors into phosphatidic acid and phosphatidylinositol, and by depletion of 32P-prelabeled polyphosphoinositides. In this report, rat granulosa cells from mature Graafian follicles were incubated with GnRH under various conditions to follow the hydrolysis of phosphoinositides and the generation of the metabolic byproducts of phospholipase C action. Granulosa cells were prelabeled for 3 h with myo[2-3H]inositol. GnRH provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol monophosphates, inositol bisphosphates, and inositol trisphosphates (IP3). Time-course studies revealed that IP3 was formed more rapidly than inositol bisphosphate and inositol monophosphate after GnRH treatment. The response to GnRH was concentration dependent (maximal at 10 ng/ml) and was prevented by a specific GnRH antagonist. Lithium chloride (1-10 mM) greatly enhanced the GnRH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. No changes were observed in the levels of free [3H] inositol and [3H]phosphatidylinositol in GnRH-treated cells. However, treatment with both lithium and GnRH for 30 min significantly reduced the levels of free [3H]inositol and [3H] phosphatidylinositol. In the presence of lithium, the rate of hormone-stimulated inositol phosphate formation was not altered by 30 min of prior treatment with GnRH, indicating that phospholipase C activity is not readily desensitized. GnRH also increased the formation of diacylglycerol (DAG), another product of phospholipase C action. In cells prelabeled with [3H] arachidonic acid, GnRH significantly increased levels of DAG in incubations lasting 2-5 min. Concomitant increases in [3H] phosphatidic acid were also observed in GnRH-treated cells. In conjunction with these studies, intracellular free Ca2+ levels were measured by Quin 2 fluorescence. GnRH and its agonistic analog rapidly increased (5 sec) cytosolic free Ca2+ levels (approximately double). The results demonstrate that an early event in the action of GnRH is the hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism. The products resulting from this action of GnRH, i.e. IP3 and DAG, may serve as intracellular mediators for the mobilization of intracellular calcium, or the activation of protein kinase C and arachidonic acid release.

Changes in the half-life of ribosomal protein messenger RNA caused by translational repression

The half-life of ribosomal protein operon L11 mRNA in vivo was measured during exponential growth by following the kinetics of incorporation of radioactive precursors into L11 mRNA transcribed from multi-copy plasmids. The degree of translational feedback regulation by L1, the L11 operon-specific translational repressor protein, was changed by altering the site on the “L11 mRNA” where L1 interacts. The half-life of the overproduced L11 mRNA increased by about fivefold when translational repression was abolished, while the half-life of mRNA from the spc ribosomal protein operon, which is not translationally regulated by L1, stayed constant. Furthermore, the half-life of L11 operon mRNA carrying an additional mutation in the ribosome binding site that abolishes translation remains short. This indicates that the change in half-life observed during increased gene dosage is due to translational repression by L1 and is probably a consequence of L1 blocking translation of L11 mRNA and not due to some nucleolytic activity mediated by L1.

GM1 Ganglioside Treatment of PC 12 Cells Stimulates Ganglioside, Glycolipid, and Lipid, but Not Glycoprotein Synthesis Independently from the Effects of Nerve Growth Factor

The incorporation of radioactive precursors into gangliosides and other glycolipids, glycoproteins, and total lipids has been studied in rat pheochromocytoma PC12 cells. Starting with the same PC12 cell pool, cultures displaying different degrees of neuritic expression in response to nerve growth factor (NGF) and combinations of serum ganglioside GM1 were produced. Attempts were then made to correlate neuritic regulation with biochemical performances of these cells. NGF stimulates the incorporation of [3H]galactose into gangliosides and other glycolipids and glycoproteins and [14C]acetate into total lipids, regardless of the serum concentration. NGF both increased their initial labeling rates and promoted additional and more extensive labeling from culture day 4 onward. Unexpectedly, exogenous GM1 also elicited an increase in ganglioside labeling as well as that of the other lipid classes, but not of glycoproteins. The GM1-induced increase was evident at higher serum concentrations (1%) regardless of the presence or absence of NGF, but not apparent in low (0.15%) serum. Serum levels themselves did not affect labeling patterns in the absence of NGF and GM1. GM1-induced stimulation of labeling reflects an increase in the synthetic activities of the cells, and not increased precursor uptake or reduced product degradation. For all constituents stimulated by GM1, concurrent treatment with NGF produces cumulative effects, suggesting independent mechanisms of action by the two molecules.

Studies of nascent lipoproteins in isolated hepatic microsomes and Golgi cell fractions

Publisher Summary This chapter reviews some of the procedures that have been used for the isolation and characterization of nascent lipoproteins from the subcellular organelles involved in plasma protein secretion obtained from liver. The emphasis is on techniques that have been useful in studies of high-density lipoproteins (HDL) synthesis and secretion by the liver of young chickens. In vivo studies, in which radiolabeled precursors of the protein and/or lipid moieties are used, are valuable in determining the types of apolipoproteins and/or lipids made by the liver. One of the major limitations of the in vivo labeling technique is obtaining sufficient incorporation of radioactive precursors into the isolated nascent lipoproteins. Despite the limitations which have been enumerated, the combination of kinetic studies, cell fractionation, and electron microscopy is an effective method in eliciting information of the intracellular sequence of events which leads to the formation of nascent lipoproteins.

Deletion of a yeast small nuclear RNA gene impairs growth.

We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10- strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non-conditional.

Effect of linear gramicidin on sporulation and intracellular ATP pools of Bacillus brevis

When Bacillus brevis ATCC 8185 was subjected to nutritional shiftdown from a rich medium to one completely devoid of a nitrogen source, sporulation could be stimulated by the addition of linear gramicidin. Gramicidin-induced sporulation occurred after a considerably longer lag period than the earlier described tyrocidine-induced process (Ristow and Paulus 1982) but involved similar associated biochemical changes, such as extracellular protease production, rapid incorporation of radioactive precursors into RNA, and dipicolinate synthesis. The increased incorporation of [3H]leucine into tyrocidine was a characteristic element in gramicidin-induced sporulation, not being observed when spore formation was accelerated by limited nitrogen supplementation. Nitrogen supplementation (0.02-0.01% nutrient broth) caused a slow and gradual increase in dipicolinate production, in contrast to the sudden, rapid rise of dipicolinate synthesis provoked by the addition of gramicidin or tyrocidine. The induction of sporulation by gramicidin occurred at very low peptide concentrations (0.03 microM), which also brought about an acute depletion of intracellular ATP. In sporulation accelerated by nutrient broth, no depression of ATP level was observed and nonionophoric analogues of gramicidin were unable to substitute for gramicidin in inducing sporulation.

Sensitivity of Preimplantation Mouse Embryos to Abrin

The effect of the plant toxin abrin on preimplantation mouse embryos in vitro was studied. Two-cell embryos from C57BL/6J or B6CBA/F1/Bom female X B6CBA/F1/Bom male mice were exposed to abrin for 72 hrs in vitro, in medium containing abrin in concentrations ranging from 0.1 to 10,000 pg/ml. Two-cell mouse embryos were also exposed to corresponding concentrations of ricin in vitro. The effect of abrin was evaluated by daily morphological observation of the embryos up to the blastocyst stage and by measuring DNA, RNA and protein synthesis by radioactive precursor incorporation. The effect of ricin was evaluated by morphological observation of the embryos up to the blastocyst stage. The results showed that 2-cell mouse embryos were highly sensitive to abrin, 1 pg/ml being the lowest concentration of the toxin that significantly inhibited the development of 2-cell embryos to the blastocyst stage. With ricin statistically significant inhibition of blastocyst formation was obtained at a concentration of 10 pg/ml. The ID50 for abrin was calculated to be 24 pg/ml and for ricin 47 pg/ml. A dose-dependent lag period was observed before the effect of abrin or ricin on the formation of blastocysts became evident. Incorporation of 3H-thymidine, 3H-uridine and 14C-leucine into DNA, RNA and protein, respectively, was also inhibited by abrin after different time intervals in culture.

Direct inhibitory actions of Gnrh on accessory reproductive organs of rat

Recently gonadotropin releasing hormone (GnRH) and its agonistic analogs were demonstrated to have some direct actions in accessory reproductive organs. In our study the effects of GnRH and its analogs on some steroid hormone induced responses were investigated. GnRH and its analogs inhibited estradiol induced ornithine decarboxylase (ODC) and glucosamine-6-phosphate synthase activities in the uterus of rat. These enzymes which are markers for cell proliferation are regulatory enzymes in the biosynthetic pathways of polyamines and glycoproteins, respectively. Similarly, GnRH and its analogs also inhibited testosterone stimulated ODC activity in ventral prostate of rat. In addition, GnRH analog inhibited incorporation of radioactive precursors into RNA and protein induced by estradiol in uterus or dihydrotestosterone (DHT) in ventral prostate. In an effort to elucidate the mechanism of action of GnRH in uterus, it was found that GnRH analog treatment does not alter the estradiol receptor content in vivo. Also, GnRH does not show any effect on radioactive estradiol binding to its receptor in vitro. Hence, the inhibitory actions of GnRH in uterus may not involve estradiol receptors. However, GnRH analogs were found to have post-transcriptional effects. It was observed that DHT induced poly(A) polymerase activity in ventral prostate and estradiol induced poly(A) polymerase activity in uterus were inhibited by GnRH analog treatment. It was further observed that GnRH inhibited incorporation of [3H]uridine into poly(A)+ RNA of ventral prostate. This indicates that the inhibitory effects of GnRH involve post-transcriptional mechanisms.