Effect of ischaemia on protein synthesis in vitro

Changes in the incorporation of 14C-amino acids into proteins in vitro were followed under conditions of ischemia induced by abdominal aorta ligature and subsequent recirculation in dogs. Cell saps isolated from L-S spinal cord, spinal ganglia, the sciatic nerve and medulla oblongata were added to the incorporation mixture composed of ribosomes and an enzymatic system from intact brains. Cytosols isolated from ischemic animals affected the rate of in vitro protein synthesis moderately, while repeated ischemia caused a profound decrease in the incorporation of amino acids into proteins. Cytosols from L-S spinal cord and especially from spinal ganglia after three days of recirculation substantially enhanced incorporation thus indicating a massive response of these tissues to ischemic injury. Cell saps from the medulla oblongata increased amino acid incorporation into proteins in vitro in all experimental groups.     

THE EFFECT OF ISCHAEMIA AND RECIRCULATION ON PROTEIN SYNTHESIS IN THE RAT BRAIN

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L-[1–¹⁴C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post-ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L-[1–¹⁴C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.
Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post-ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.     

Influence of blood sera from dogs subjected to ischemia and recirculation on incorporation of14C-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.     

Synthesis of proteins in heart mitochondria during postnatal development of the rat

Proteins of inner mitochondrial membranes of the albino rat myocardium during postnatal development of 1, 3 and 6 months old animals were electrophoretically separated in 10% polyacrylamide gel. The rate of 14C-amino acids incorporation into examined proteins was determined in vitro. Specific radioactivity of the total mitochondrial fraction decreased in the course of the postnatal development. That of outer membranes remained unchanged, though it sharply increased in inner membranes of mature animals as compared with animals aged one month. Levels of radioactive precursor incorporation in separate protein fractions of inner membranes of the myocardium mitochondria were estimated.     

Synthesis of a Stress Protein Following Transient Ischemia in the Gerbil

In vitro translation products of gerbil brain preparations, obtained from animals killed during recirculation following transient ischemia, showed increased synthesis of a 70-kilodalton stress protein, identified by two-dimensional gel electrophoresis. Stimulation of stress protein synthesis was evident as early as 2 h after recirculation, at which time overall translation activity remained low. Expression of the 70-kilodalton protein reached a maximum at 8 h recirculation, when incorporation into other translation products had returned to essentially control levels. Increased incorporation into the stress protein was still detectable after 24 h recirculation. Although the functional consequences of increased expression of this stress protein remain unknown, these results suggest that the gerbil ischemia model may provide a useful experimental system in which to study the involvement of this phenomenon in processes related to postischemic cell damage and recovery.     

Effect of ischaemia on protein synthesis in neuron and glia-enriched fractions from the rabbit spinal cord

The incorporation of 14C-leucine into the post-mitochondrial supernatant and neuron, glia and myelin-enriched fractions isolated from the rabbit spinal cord was studied after ischaemia and subsequent recirculation. In the cell-free system, incorporation decreased to 55% of the control value after 40 min ischaemia, but proteosynthesis returned to the pre-ischaemic value after 3 h recirculation and remained at this level during further recirculation. The incorporation of amino acids into proteins of neurons and neuroglia differed from the cell-free system and from each other. In the enriched neuronal fraction, protein synthesis fall after ischaemia and also during the first hours of recirculation, but during further recirculation it rose to 60% above the control value. In the enriched glial fraction, specific radioactivity of proteins rose abruptly immediately after ischaemia and by the fourth day there was sixfold increase as compared with control values. The results indicate that the ischaemia-induced decrease in protein synthesis is only transient and that a significant increase occurs in the surviving cell populations, especially the neuroglia. The functional changes caused by spinal cord ischaemia are irreversible, however.     

Some biochemical effects of insulin and steroid hormones on the rat prostate in organ culture

The effect of various steroids and insulin on expiants of the ventral prostate of adult rats in organ culture was investigated. Testosterone activated the incorporation of labeled precursors into RNA and protein and the formation of ¹⁴CO₂ from ¹⁴C-glucose by expiants. All these effects were mimiced by insulin. The testosterone action was suppressed by cyproterone in vitro. In addition to androgens, glucocorticoids activated the incorporation of ³H-uridine into RNA. Simultaneous addition of testosterone with hydrocortisone or insulin produced an augmented effect on the incorporation of ³H-uridine into RNA which exceeded that resulting from a simple summation of the individual hormone responses. Insulin facilitated likewise the action of testosterone on the incorporation of ¹⁴C-amino acids into protein. When all three hormones were added simultaneously to the culture medium the stimulation of the three biochemical parameters was maximal. It was therefore concluded that all three hormones are necessary for an adequate maintenance of the ventral prostate of the rat.     

Crystallin synthesis in the lens of newborn mice]

Lenses of newborn mice were incubated for different time in the Hanks solution containing 14C-amino acids mixture. Syntheses of gamma-crystallin and subunits of alpha-crystallin were shown to start at the first minute of the incybation. The incorporation rate of 14C-amino acids into gamma-crystallin was twice as high as that into alpha-crystallin within 5 minutes of the incubation. The assembly of alpha-crystallin tetramers took place after 5 minutes from the beginning of the incubation. Preincubation with actinomycin D for 3 and 6 hours resulted in the decrease of 14C-amino acids incorporation into gamma-crystallin only. These data suggest that the synthesis of gamma-crystallin takes place on both short-lived and long-lived mRNAs. Alpha-Crystallin subunits are supposed to synthesize only on long-lived mRNAs.     

Paragonial proteins of Drosophila melanogaster adult male: In vitro biosynthesis

When paragonia were incubated for 8 hr in simplified Robb's medium a continuous increase in incorporation of ¹⁴C-amino acids into TCA precipitable proteins was found. The increase is linear over the first 2 hr.Copulation provides the stimulus for an alteration in rate of in vitro incorporation: a rapid and clear increase occurs. A similar increase in rate of incorporation of ¹⁴C-uridine into RNA and ³H-ethanolamine into ‘paragonial substance’ could be found. The pattern of incorporation of ¹⁴C-amino acids into proteins, as determined by polyacrylamide gel electrophoresis, remains qualitatively unaltered. Results from transplantation experiments of single paragonia suggest that the stimulation, at least in part, is neuronal or humoral mediated.In vitro incubations with actinomycin D, under conditions of 90% inhibition, resulted in an increased rate of incorporation of ¹⁴C-amino acids into proteins.     

Dependence of inhibition by levorin of amino acid incorporation into protoplasts and subcellular proteins of Candida albicans on the change of endocellular potassium concentration]

Levorin is found to decrease more efficiently potassium concentration in C. albicans protoplasts under their incubation in the presence of sodium than in the medium containing the equivalent amount of potassium. Minimal inhibitory concentration of levorin for resistant C. albicans cells incubated on potassium-depeleted medium was in 4 times lower than for cells incubated in potassium-enriched medium. The decrease of membrane permeability for 14C-amino acids and their incorporation into membrane, ribosomal and soluble proteins under the effect of levorin was more pronounced when protoplasts were cultivated in sodium-containing medium than in potassium-containing one. In both media the inhibition of 14C-amino acid incorporation by levorin into ribosomal and cytosol proteins was more efficient than into membrane proteins, but these differences were less pronounced in case of potassium-containing medium.     

Effects of salicylic acid on protein content and <Superscript>14</Superscript>C-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.     

Effect of novoimanine on the cellular permeability indices of staphylococci]

Novoimanine is an antibacterial drug from Hypericum perforatum L. When used in the bacteriostatic concentration, i.e. 0.5 gamma/ml, it induced release of potassium ions from the cells of Staphylococcus aureus 209P and had no effect on release of the UV-absorbing compounds and 14C-amino acids. In addition, incubation of the cells with novoimanine (2.5--50 gamma/ml) provided "preservation" in them of the earlier absorbed 14C-amino acids, while in the control cells their level decreased. In a concentration of 100 gamma/ml novoimanine stimulated activity of ATP-ase and alkaline phosphatase by 34 and 37-57 per cent respectively. Histones F1 and F3 of the calf thymus induced an intensive release of 14C-amino acids from the cells of staphylococci and increased the activity of ATP-ase by 6-10 times. The data of the study suggested that the effect of novoimanine on the cytoplasmic membrane was limited and different from that on the polycationic antibacterial agents.     

Amino acid metabolism and protein synthesis in streptomycin resistant Vibrio El Tor.

Metabolic activities in relation to protein synthesis and amino acid utilization are altered in Vibrio El Tor after development of resistance towards streptomycin. Efficiency of in vivo and in vitro protein synthesis is markedly reduced in streptomycin resistant Vibrio El Tor. The rate of incorporation of 14C-amino acids into protein, uptake of 14C-valine and oxidation of certain amino acids are also altered.     

The effect of castration on the ribonucleic acid metabolism of an experimental prostatic tumour

1. The incorporation of [(14)C]phenylalanine into the protein of microsomes obtained from an androgen-dependent transplantable prostatic tumour has been investigated. 2. The incorporation of [(14)C]ATP into the RNA of the nuclei from the same tumour has also been examined. 3. The castration of the host animal depresses the incorporation of both labelled compounds on subsequent incubation in vitro. 4. The uptake of phenylalanine can be markedly stimulated by polyuridylic acid only in tumours obtained from castrated host animals. 5. The tumour RNA-polymerase activity appears to be dependent on the concentrations of androgens in the host animal.     

Conversion of 14C-galactose into amino acids in tissue culture cells and its inhibition by manganese

The incorporation of 14C-galactose into primary AGMK-cells was studied in the presence and absence of Mn2+. The transport of galactose into the cells is not influenced by Mn2+. 1 mM MnCl2 inhibits the incorporation of galactose into acid-precipitable material up to 50% after 6 hours incubation. In the absence of Mn2+ a substantial amount of galactose is converted to glucose, which is mainly metabolized into aspartic acid and serine. The conversion of galactose into glucose is inhibited by the addition of Mn2+. However, Mn2+ does not influence the activity of the UDP-galactose-4'-epimerase in vitro. Using the SDS-polyacrylamide electrophoresis the labelling of protein bands is similar with 14C-galactose or a 14C-amino acid mixture, respectively. In the presence of Mn2+ the incorporation of both galactose or amino acids is inhibited: With amino acids the inhibition is observed in all protein bands, whereas with galactose some bands remain unaffected. It is concluded that these are galactoproteins.     

Effect of experimental diabetes on the incorporation of amino acids into protein in rat aorta.

The incorporation of 14C-labelled leucine or phenylalanine into alkali-soluble protein was determined under in vitro conditions in aortic intima-media of normal and streptozotocin-diabetic rats. Two weeks after the induction of diabetes the incorporation of the amino acids into aortic protein was reduced. When determined after diabetes of one week's duration the leucine-14C incorporation was not significantly reduced, while after 5 weeks of diabetes it was severely impaired. After administration of insulin to diabetic rats in vivo for 2 weeks there was no difference in leucine-14C incorporation between normal and diabetic rats. Addition of insulin (0.1 U/ml) in vitro had no effect on the leucine-14C incorporation in either normal or diabetic aorta during incubation times of 3 or 6 h. Elevation of the glucose concentration in vitro from 5.6 to 22.2 mmol/l did not influence the leucine incorporation in diabetic aorta. Both the aortic wet weight and the aortic content of alkali-soluble protein were decreased after 5 weeks of diabetes. The decrease in the protein content of aorta of diabetic animals suggest that the protein synthesis is impaired in vivo.     

Plasma orosomucoid metabolism and susceptibility to malarial infection in rodents

The effects of Plasmodium berghei infection on liver function and plasma orosomucoid metabolism were investigated in Wistar rats. Infected rats with 20-25% parasitaemia manifested increased serum transaminase levels, hypoalbuminaemia and hypoproteinaemia. In spite of such indications of deranged liver function, the hepatic synthesis rate (as measured by 14C-amino acid incorporation) of seromucoids predominantly orosomucoid or alpha 1-acid glycoprotein) was increased by 73%. The circulating levels of this glycoprotein were also doubled in infected animals. The albumin synthesis rate was not increased. This preferential synthesis and increase in circulating levels of orosomucoid may have in vivo significance in malarial infection, in view of reports that orosomuocid has influence on in vitro invasion of red cells by malarial parasites.     

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.     

Changes in Amino Acid Metabolism in vivo with Time after Feeding

Changes in the metabolism in vivo of amino acids with the lapse of time after feeding a diet were investigated by measuring the incorporation of ¹⁴C into some body components one hour after injection with ¹⁴C-amino acid mixture.

The incorporation of ¹⁴C into protein in the liver and carcass was rather constant, but that into blood sugar, liver glycogen, and lipids in the liver and carcass showed a change with the lapse of time after feeding a 25% casein diet or a protein-free diet. The incorporation of ¹⁴C into liver glycogen was stimulated shortly after feeding, but it was reduced at 7 hr, when a large amount of glycogen was still in the liver. On the contrary, the specific activity of blood sugar increased with the lapse of time after feeding. The conversion of ¹⁴C-amino acids into lipids in the liver and carcass was stimulated shortly after feeding.

The incorporation of ¹⁴C into protein was higher in the rats fed the protein-free diet than in those fed the 25% casein diet, and the higher incorporation was partly counterbalanced by the lower incorporation of ¹⁴C into lipids and glycogen in the rats fed the protein-free diet.     

Effects of the rat renotropic system on 14C-uridine incorporation into RNA and RNA precursors.

We used the incorporation of ¹⁴C-uridine into RNA of incubating kidney fragments from normal control rats to evaluate RNA metabolism. Sera from unilaterally nephrectomized rats (uni) obtained 20 hrs post-operatively stimulate ¹⁴C-uridine incorporation into RNA significantly more than sera from sham-operated rats (sham). Differently, sera from uni and sham rats have little influence on specific activities of endogenous uridine nucleotide pool in renal fragments. Renal extracts were obtained by homogenizing kidneys in saline. Extracts from kidneys of uni and sham rats 20 hrs post operation depress incorporation markedly, and each depresses to a similar extent, but kidney extracts dilute the specific activities of uridine pools. Correcting for the latter dilution demonstrates that kidney extracts alone have little effect on ¹⁴C-uridine incorporation into RNA. We then followed the results when these sera and extracts were combined. Compared to fragments incubating in sham sera and sham extracts, substitution of uni extracts or both uni extracts and uni sera enhances ¹⁴C-uridine into renal RNA, whether or not results are corrected for changes in the specific activities of the uridine pools. We conclude that after uninephrectomy there is a concurrent elevation in circulating renotropin and a tissue activating factor in the remaining kidney. The tissue factor can only form an excitor to ¹⁴C-uridine incorporation into RNA when serum is present. The rat renotropic system that enhances incorporation of ³H-thymidine into DNA also can stimulate ¹⁴C-uridine incorporation into renal RNA.