[14C]Formate and [8-14C]Adenine incorporation into purine nucleotides of ischaemic rat liver after re-establishment of blood flow

• 1.1. After restoration of blood flow the incorporation rate of [¹⁴C]formate and [8-¹⁴C]adenine into purine nucleotides of ischaemic rat liver was investigated.
• 2.2. The incorporation rate of [¹⁴C]formate into adenine nucleotides was nearly 5-fold greater in post-ischaemic livers than in controls at 120 min point following blood restoration.
• 3.3. No difference in the rate of [8-¹⁴C]adenine incorporation into adenine nucleotides of post-ischaemic and control rat liver lobes was found.
• 4.4. After re-establishment of blood circulation a predominant contribution of the de novo biosynthetic pathway in the recovery of purine nucleotides of the ischaemic rat liver is emphasized.

     

Studies of the metabolic consequences of underperfusion in the isolated rat liver using a once-through perfusion technique

• 1.1. Interruption of the flow of medium to the isolated perfused rat liver results in a more reduced redox state for both the cytoplasmic and mitochondrial compartments, together with a loss of adenine nucleotides associated with a decreased content of ATP and increased concentrations of ADP and AMP.
• 2.2. Restoration of the flow reverses many of these changes.
• 3.3. The period of reperfusion is associated with increased rates of urea formation and gluconeogenesis.

     

Effect of inorganic phosphate on synthesis of 5-phosphoribosyl-1-pyrophosphate in cultured plant cells

• 1.1. Inorganic phosphate (Pi) was absorbed rapidly by suspension-cultured cells of Catharanthus roseus which had previously been cultured in Pi-free Murashige Skoog medium.
• 2.2. The intracellular levels of ATP, ADP and 5-phosphoribosyl-l-pyrophosphate (PRPP) increased markedly during the 24 hr which followed the addition of Pi (1.25mM).
• 3.3. Availability of PRPP in vivo, estimated by the measurement of nucleotide synthesis from [8-¹⁴C]adenine, was also increased by addition of Pi.
• 4.4. Only a 20% increase in the maximum catalytic activity of PRPP synthetase was observed in extracts of cells, prepared 24 hr after addition of Pi.
• 5.5. In contrast to results for mammalian PRPP synthetase, the activity of PRPP synthetase, partially purified from Catharanthus roseus, was inhibited by concentration of Pi greater than 5mM.
• 6.6. The mechanisms involved in the increased availability of PRPP and the synthesis of adenine nucleotides in the plant cells cultured in Pi-containing medium are discussed.

     

Specificity of transcription of single-copy DNA in different rat tissues

• 1.1. The amount of single-copy DNA sequences transcribed in normal tissues of adult rats (brain and liver) and in the Guerin ascites tumor (GAT) was determined by hybridization of in vitro labelled ¹²⁵I-single-copy rat DNA with a vast excess of total nuclear RNA to very high Rot values (up to 350,000).
• 2.2. The tissue specificity of total nuclear RNA (nRNA) was estimated by annealing of single-copy DNA to a mixture of nuclear RNAs of two different organs (brain + GAT; liver + GAT).
• 3.3. Liver and GAT RNAs annealed to about 12% of the single-copy DNA.
• 4.4. Hybridization with a mixture of the two RNAs increased slightly the amount of hybridization.
• 5.5. In contrast to other tissues, brain nuclear RNA hybridized to a much higher level (20% of the single copy DNA). Addition of GAT RNA did not increase this value.

     

Effect of conjugation on the incorporation of glycine into Tetrahymena

• 1.1. Conjugation of Tetrahymena enhanced the incorporation of glycine into the nuclear fraction by 500%.
• 2.2. Incorporation of glycine into the microsomal supernatant was augmented by almost 500% by conjugation.
• 3.3. Mitochondrial incorporation was stimulated nearly 3-fold in the conjugating strains while the incorporation of glycine into the microsomes was enhanced approximately 2.5 times.
• 4.4. In the whole cell, glycine incorporation was increased nearly 2-fold by conjugation.
• 5.5. Strong nuclear involvement was indicated by elevated metabolic activity and incorporation of glycine into RNA and DNA.
• 6.6. Stimulation of the metabolism of Tetrahymena by cell communication suggests that the contents of a cell can have a synergistic effect on another cell.
• 7.7. Augmentation of the biosynthetic capacities of cells by fusion is a demonstration of the dominant role of the cell membrane in the regulation and control of cells.
• 8.8. Enhancement of biosynthesis of nuclear proteins in conjugating strains of cells indicates that fusion gives rise to the synthesis of new protein from previously existing protein or protein procursors.
• 9.9. The specific activities of the subcellular fractions after the incorporation of glycine into 2 separated starved strains of Tetrahymena followed the usual pattern of nucleus less than whole cells, whole cells less than mitochondria, mitochondria less than microsomes, but with the microsomal supernatant being much greater than that of the microsomes.

     

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. II. Effects of adenine nucleotides

• 1.1. The native rat-kidney cortex Fructose-1,6-bisphosphatase is differentially regulated by adenine nucleotides in the presence of divalent cations.
• 2.2. Binding of AMP and ADP to the enzyme is co-operative. The inhibition by both nucleotides show an uncompetitive mechanism AMP being the most efficient inhibitor.
• 3.3. Mg²⁺ decreases the inhibition produced by AMP and ADP by enhancing their I_0.5 and completely annulates the inhibitory effect of ATP.
• 4.4. In the presence of Mn²⁺ ADP behaves as an inhibitor but no inhibition is evident with AMP, suggesting the existence of different allosteric sites for each nucleotide.

     

Energy metabolism of human erythrocytes in psoriasis

• 1.1. Adenine nucleotide concentrations and metabolism in red blood cells (RBC)2 and RBC ghosts from psoriatic patients and healthy subjects were compared.
• 2.2. The ATP and total adenine nucleotide levels and the adenylate energy charge (EC) were elevated in the blood from psoriatic patients.
• 3.3. The rate of glycolytic production of ATP by intact RBC was unchanged, but the Na⁺, K⁺-ATPase activity of RBC ghosts was decreased significantly in psoriasis.
• 4.4. Results suggest that the defect in adenine nucleotide metabolism is a systemic manifestation of psoriasis, and that the quantification of adenine nucleotides in RBC and in whole blood samples may be of pathophysiological value in psoriatic lesion.

     

Contrast in adenine uptake by chicken and rabbit erythrocytes in vitro

• 1.1. Relative to rabbit erythrocytes, chicken red blood cells exhibit a much greater capacity to utilize [³H]adenine for nucleotide synthesis in vitro, even at 5°C and in the absence of added inorganic phosphate.
• 2.2. This difference is largely due to a higher concentration of phosphoribosylpyrophosphate and greater activity of adenine phosphoribosyltransferase in the avian cells. lli]3. The capacity of avian erythrocytes for utilization of guanine and hypoxanthine is several fold less than that of adenine.
• 3.4. The data are consistent with lower activity for hypoxanthine/guanine phosphoribosyltransferase than for adenine phosphoribosyltransferase in intact chicken erythrocytes.
• 4.5. The results indicate that reutilization of adenine by chicken erythrocytes may be physiologically significant.

     

Studies on compartmentation of uridine 5'-triphosphate during synthesis of cytoplasmic and plastid ribosomal RNA's in Euglena gracilis

• 1.1. Compartmentation of uridine 5'-triphosphate (UTP) was studied during synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and plastid ribosomal RNA (pl-rRNA) in photoorganotrophically grown cells of Euglena gracilis Z.
• 2.2. Using the approach of Wiegers et al. (1976) the steady state specific radioactivity of UTP was compared with that ofcyt-20S rRNA, cyt-25S rRNA, pl-16S rRNA and pl-23S rRNA under low and at 100-fold higher specific radioactivity of exogenously fed pHl-uracil.
• 3.3. The equal steady state specific radioactivities of all rRNAs at both feeding conditions argue against compartmentation of UTP during their synthesis.
• 4.4. At high specific radioactivity of exogenous [³H]-uracil the salvage-derived labelled UMP was shown to be diluted 15,000-fold by unlabelled UMP formed de novo, whereas this dilution factor was 100-fold lower at low specific radioactivity of [³H]-uracil indicating inhibition of the de novo synthesis of UMP.
• 5.5. Transport is suggested of uridine nucleotides into chloroplasts by the 15-fold higher specific radioactivity of intracellular [³H]-uracil than that of UTP as well as UMP residues in pl-rRNA.

     

Age-related changes in total dna and rna and incorporation of uridine and thymidine in rat liver,kidney and spleen

• 1.1. Total content of DNA and RNA in liver, kidney and spleen were measured in young and aged rats. At the same time the incorporation of [¹⁴C]thymidine, a DNA precursor, and [³H]uridine, an RNA precursor, were also determined.
• 2.2. Changes in total organ DNA and RNA correlated with sexual maturation as did incorporation of precursors.
• 3.3. Young animals have more DNA per organ relative to RNA. with kidney and spleen DNA showing a decrease between maturity and senescence.
• 4.4. However, liver RNA increases with age. a change probably due to decreased catabolism of RNA since [³H]uridine uptake decreases.
• 5.5. Liver polyploid differentiation, and [¹⁴C]thymidine and [³H]uridine uptake, are correlated.
• 6.6. In kidney, incorporation of [³H]uridine is inversely related to [¹⁴C]thymidine incorporation.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

Effects of adenine nucleotides on low Km 5′ nucleotidase from human seminal plasma

• 1.1. Low K_m 5' nucleotidase purified from human seminal plasma has been used in this study to investigate the response of the enzyme to adenine nucleoside di- and triphosphates in the presence of AMP and IMP as substrates.
• 2.2. In the presence of AMP, the addition of 0.5 mM ATP to the enzyme Mg-free results into the highest V_max/K_m ratio value and other experimental combinations of effectors tested cause variation of the kinetic parameters of the enzyme, indicating a control of AMP dephosphorylation by adenine nucleotides.
• 3.3. In the presence of IMP, ATP and ADP activate the enzyme but the response to various experimental combinations of effectors shows no significant difference in the kinetic properties of the enzyme, indicating a different control of the dephosphorylation of IMP.

     

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose,hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

• 1.1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile.
• 2.2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue).
• 3.3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose.
• 4.4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus.
• 5.5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

     

A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

• 1.1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves.
• 2.2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography.
• 3.3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA.
• 4.4. The three enzymes require either Mg²⁺ or Mn²⁺ for activity.
• 5.5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase.
• 6.6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.

     

Cytosolic glutamate dehydrogenase in adult Hymenolepis diminuta (cestoda)

• 1.1. Glutamate dehydrogenase (E.C. 1.4.1.2) isolated from anterior proglottids of the rat tapeworm, Hymenolepis diminuta is found primarily in the cytoplasmic fraction.
• 2.2. The enzyme is NAD-specific and markedly affected by assay pH. Decreasing the assay pH from 7.4 to 6.5 reduces the apparent K_m for α-ketoglutarate about 10-fold, while under similar conditions, the apparent K_m for glutamate remains unchanged. In addition, at low substrate concentrations, NADH oxidation is favored.
• 3.3. The nucleotides AMP, ADP, ATP, GDP and GTP have a little effect on glutamate dehydrogenase (GDH) activity. A possible physiological role for the cytoplasmic GDH in Hymenolepis diminuta is discussed.

     

A comparative study of the basic nuclear proteins from sperm of bivalve molluscs

• 1.1. Basic nuclear proteins from spermatozoa of the three mollusc species belonging to the class Bivalvia have been analyzed using one- and two-dimensional electrophoresis.
• 2.2. Four nuclear basic proteins have been purified and their amino acid compositions determined.
• 3.3. In these spermatozoa histone-type proteins coexist with protamine-like proteins.
• 4.4. The protamine-like proteins that have been studied show different electrophoretic behavior but in general are similar, with a high content of lysine, arginine, alanine and serine.
• 5.5. Interspecific variability has been found for the H1-like histone.

     

Anuran amphibia which are not acclimable to high salt,tolerate high plasma urea

• 1.1. The capacity of five anuran Amphibians (Bufo viridis B. regularis, Rana ridibunda, Hyla arborea and Pelobates syriacus) to acclimate to NaCl and urea solutions was investigated.
• 2.2. All species could be acclimated to relatively high concentrations of urea solutions, while only Bufo viridis and Hyla arborea could be acclimated to 500 mOsm/kg or higher NaCl solutions.
• 3.3. The plasma urea concentration in B. viridis and H. arborea was elevated to levels over 140 mmol/1.
• 4.4. The sum of plasma sodium and chloride concentrations did not increase over 400 mmol/l in any species.
• 5.5. Urine osmolality, which was normally low, increased, but never exceeded the plasma osmolality.
• 6.6. In the urea acclimation conditions, urine electrolytes diminished, similarly in all species in this study.
• 7.7. It is concluded that anuran Amphibians can tolerate high plasma urea concentrations, but only those species which can elevate it, either through retention or net synthesis, can be acclimated to high salt solutions.

     

Effect of adenosine on gluconeogenesis in the perfused liver of chicken and guinea-pig

• 1.1. Glucose formation from lactate by the perfused liver of 48 hr starved chickens was strongly inhibited by adenosine (Ado); the half-maximal inhibition was attained at 40 μM. This effect was paralleled by a four- to five-fold increase of ATP content as determined in freeze-clamped liver.
• 2.2. In chicken liver homogenate gluconeogenesis from precursors such as alanine, glutamate, glutamine and aspartate, which are not converted into glucose by the perfused chicken liver, proceeded at rates equal to or higher than that with lactate, being markedly inhibited by Ado.
• 3.3. In the perfused guinea-pig liver glucose synthesis with lactate, propionate, glycerol and fructose was also inhibited by Ado; however, when precursors such as pyruvate, glutamine and a mixture of lactate + pyruvate were supplied to the liver Ado did not inhibit gluconeogenesis.
• 4.4. Assay of adenine nucleotides in the perfused guinea-pig liver, stopped by freeze-clamping technique in a number of experimental variants, revealed no correlation between the rate of gluconeogenesis and the changes induced by Ado in the adenine nucleotide pool.
• 5.5. In the perfused liver of both chicken and guinea-pig Ado produced an increase of the lactate to pyruvate ratio and, in general, a diminution of the content of malate-aspartate shuttle intermediates.
• 6.6. The results are interpreted as suggesting that the inhibitory effect of Ado on hepatic gluconeogenesis is not necessarily mediated by the changes in the adenine nucleotide pool.