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.

     

Light-induced transient increase of the activity of topoisomerase I in plasmodia of Physarum polycephalum

• 1.1. Activity of topoisomerase I and incorporation of [³H]uridine and [¹⁴C]thymidine were monitored during light-induced sporulation of the slime mold Physarum polycephalun.
• 2.2. A 4-fold transient increase of topoisomerase I activity but not of [³H]uridine or [¹⁴C]thymidine incorporation was observed after 42 hr of illumination with 6 hr impulses.
• 3.3. The activity of topoisomerase I did not increase in the absence of light impulses. However, ca 5-fold increase of the activity was observed in dark when 100 μ M dibutyryl-cAMP was administered 12 hr before harvesting of plasmodia.
• 4.4. Fluorodeoxyuridine and cycloheximide administered 36 hr after starting of the illumination cancelled the increase of the activity of topoisomerase I.
• 5.5. After 7 days of the illumination, when fruiting bodies appeared, the activity of topoisomerase I dropped to about 15% of the initial value.

     

In vitro substrate utilization for lipid synthesis in liver explants from hyperthyroid chickens

• 1.1. Indian River male broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 18, 24 and 30% protein + 0 or 1 mg triiodothyronine (T₃)/kg of diet to study energetic costs of lipogenesis and the use of various substrates for in vitro lipogenesis.
• 2.2. De novo lipid and CO₂ production were determined in the presence of [1-¹⁴C]pyruvate, [2-¹⁴q]pyruvate, [3-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine.
• 3.3. Oxygen consumption was determined in mitochondrial preparations to estimate the energetic costs in expiants synthesizing lipid.
• 4.4. Radiolabeled CO₂ derived from [1-¹⁴C]pyruvate was used as an estimate of coenzyme A availability in liver expiants. Lipids derived from [2-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine estimate relative substrate efficiency.
• 5.5. Labeled CO₂ production from [1-¹⁴C]pyruvate was greatest in that group fed a 12% protein diet and least in the group fed a 30% protein diet.
• 6.6. In addition, T₃ increased CO₂ production from [1-¹⁴C]pyruvate.
• 7.7. The production of ¹⁴CO₂ from the second carbon of pyruvate or acetate was increased by T₃.
• 8.8. The low-protein diet (12% protein) increased (P <0.05) lipogenesis.
• 9.9. Adding T₃ to the diets decreased carbon flux into lipid from all substrates, but increased CO₂ production from all substrates without changing stage 3 and 4 respiration rates in mitochondrial preparations.
• 10.10. These observations imply that coenzyme A availability may have regulated de novo lipogenesis in the present study.
• 11.11. It was also concluded that previously noted effects of T₃ on intermediary metabolism may involve metabolic pathways that do not involve changes in mitochondrial function.

     

Purine metabolism in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) Seedlings

• 1.1. The metabolism of purine bases and nucleosides in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) was studied.
• 2.2. A large portion of absorbed [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]adenosine was salvaged in nucleotide and nucleic acid synthesis.
• 3.3. Most of the radioactivity of [8-¹⁴C]hypoxanthine and [8-¹⁴C]inosine was incorporated into allantoin and allantoic acid.
• 4.4. Activity of adenine phosphoribosyltransferase in enzyme extracts was much higher than that of hypoxanthme and guanine phosphoribosyltransferase(s).
• 5.5. Apparent activity of adenosine kinase was higher than that of inosine kinase. 6. NAD⁺-dependent xan thine dehydrogenase was detected in both cotyledons and embryonic axes of the seedlings.
• 6.7. The capacity of purine salvage was higher m 24 hr old cotyledons than 24 and 48 hr old embryonic axes. The reverse was observed concerning that of purine degradation.

     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

The assembly of the DNA complex present in chick embryo cell cytosol

• 1.1. Chromatography of chick embryo fibrobast cytosol labelled with [³H]thymidine or [³H]uridine precursors has shown the presence of early labelled DNA and RNA eluting at a position corresponding to a relative molecular mass of approximately 1.5–10⁵.
• 2.2. The early DNA-RNA (heteroduplex?) then moves progressively to a higher molecular weight peak, relative molecular mass approximately 10⁶.
• 3.3. The process appears similar in cytosol from cultured cells and from whole aminiotically labelled chick embryo: consequently the cytosolic DNA complex is not an artefact of cell culturing.
• 4.4. The relative contribution of artefactual and specific cytosol-associated DNA material is discussed: it is concluded that while both are present in cytosol as prepared, it is possible to discriminate between specific and artefactual DNA material.

     

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.

     

Potassium contracture of anterior byssus retractor muscle of Mytilus edulis

• 1.1. The potassium contracture in the anterior byssus retractor muscle of Mytilus edulis relaxed spontaneously, and the relaxation was accelerated by 5-HT (10⁻⁶ M), but the contractile activation and inactivation was not affected significantly.
• 2.2. By reducing [Ca]₀, the “activation curve” was shifted downward at higher [K]₀, and the “inactivation curve” was shifted toward lower [K]₀ and the rate of inactivation was increased.
• 3.3. The steady-state inactivation was maintained for at least 2 hr without complete inactivation.
• 4.4. The half-inactivation time was dependent on [K]₀, while the half-relaxation time in the contracture induced by conditioning K solutions was not.

     

Synaptic transmission at high pressure: Effects of [Ca2+]o

• 1.1. The effects of pressure on synaptic currents were examined in crayfish abdominal muscles.
• 2.2. Helium pressure (10.1 MPa) considerably decreased extracellulariy-recorded excitatory junctional potentials associated with increased short-term facilitation.
• 3.3. These effects could be mimicked by a reduction of [Ca²⁺]_o, and partially compensated by an increase in [Ca²⁺]_o.
• 4.4. Pressure also reduced the amplitude of the extracellular nerve terminal potentials (ENTP) by up to 25%, and significantly increased synaptic delay in a [Ca²⁺]_o-dependent manner.
• 5.5. The interaction between compression and various [Ca²⁺]_o were analysed in terms of an existing model of transmitter release. The results were consistent with the hypothesis that high pressure decreases the maximal Ca²⁺ influx into nerve terminals.
• 6.6. The decreased ENTP and increased synaptic delay suggest that additional processes may be involved in pressure effects on synaptic transmission.

     

The effect of temperature on the acid-base status of the blood of the hatching quail (Coturnix c. japonica)

• 1.1. The ambient temperature of embryos of pipped eggs was reduced from 38 to 28°C for a period of 45 min.
• 2.2. The blood P_CO2 was lower and the blood more alkaline at 28°C than at 38°C.
• 3.3. At 28°C plasma [HCO₃⁻] ] was lower than predicted from the blood buffer line determined in vitro.
• 4.4. The plasma concentrations of strong ions and lactate were the same at both temperatures.
• 5.5. After the ambient temperature had been returned to 38°C for a period of 45 min, blood pH was more acidic than before cooling, but there was no difference in blood P_CO2.
• 6.6. The plasma [HCO₃⁻] was the same as that at 28°C and plasma [K⁺] was higher than before cooling.
• 7.7. The results arc discussed in relation to the factors affecting blood pH in embryos at this stage of development.

     

The effect of orotic acid treatment on the energy and carbohydrate metabolism of the hypertrophying rat heart

• 1.1. Adenine nucleotide concentrations in normal and one day hypertrophied hearts of untreated, orotic acid (OA), uridine, uracil, dihydroorotate and reserpine pretreated rats were measured. OA treatment increased the ADP concentration 5-fold in one day hypertrophied hearts. Neither uracil, uridine, dihydroorotate nor reserpine treatments changed ADP or total adenylate concentrations at one day of hypertrophy.
• 2.2. The adenine nucleotide ratio (ANR) at 0.263 × 10³ M⁻¹ and the energy charge (0.66) were at their lowest values in OA and in reserpine treated one day hypertrophying hearts. The temporal decline in the indices of energy metabolism corresponded with the OA induced maximum stimulation of contractility and maximum rates of protein, RNA and glycogen synthesis.
• 3.3. The phosphorylation state of the adenine nucleotides (PSAN) was both the most sensitive and the best predictive index of the cellular energy status in normal and hypertrophying hearts. The pronounced ability of OA treatment to energize myocyte cytoplasm was shown by the 9- and 6-fold greater values of PSAN over ANR in one and three day hypertrophied hearts. The enhanced PSAN may be the key factor in the mechanism of OA induced enhancement of contractile and synthetic functions of the heart in compensatory hypertrophy.
• 4.4. The development of myocardial hypertrophy in untreated rats resulted in a 36% reduction in the cytoplasmic NAD/NADH ratio. In rats treated with OA this redox couple of the hypertrophying heart was more oxidized and was increased by 30% to restore it to the value range of normal heart.
• 5.5. The regulatory status of the glycolytic pathway in untreated and OA treated hypertrophying hearts was assessed by comparisons of the mass action ratio (MAR) and equilibrium constants for each of the individual glycolytic reactions. There was an OA induced 2.7-fold increase in glycogen, UDP-glucose and total uridine nucleotides in hypertrophied hearts. The concentrations of seven out of ten glycolytic intermediates, including pyruvate and lactate were increased as a consequence of OA treated hypertrophy. Glycolytic flux was not stalled, rather the pathway was “more open” permitting greater throughput of intermediates with individually increased levels of selected metabolites. OA stimulated hypertrophy did not change the canonical control of glycolysis by the activities and individual MAR values of phosphofructokinase and pyruvic kinase.
• 6.6. Elevated levels of Glu 6-P, Fru 6-P and DHAP can force glycolytic intermediate entry into the non-oxidative reaction segment of the pentose pathway (PP), thereby elevating Rib 5-P concentration by reversal of the conventional flux direction of PP. Rib 5-P is rate limiting for PRPP and nucleotide synthesis and increases in its concentration in OA treated hypertrophying hearts can inter alia explain the elevation in adenylate concentrations.
• 7.7. OA does not act directly on the isolated normal or hypertrophying heart neither as an inotropic agent nor as a metabolic substrate. Its myocardial action requires whole-body integration and its principal metabolic fate involves the liver and the activation of the salvage pathway of pyrimidine biosynthesis. Preformed bases and nucleosides, mostly as uridine, enter and enhance the domains of pyrimidine, purine nucleotide and RNA metabolism in the OA stimulated hypertrophying myocardium.

     

Adenosine and deoxyadenosine metabolism in lymphocytes from horses,Equis cabalus

• 1.1. The toxic effects of adenosine and deoxyadenosine on lymphocytes from horses were evaluated.
• 2.2. Mitogen-stimulated peripheral blood lymphocytes (PBL) were found to be more sensitive to the inhibitory effects of adenosine than were lymphocytes from spleen, lymph node and thymus.
• 3.3. Adenosine deaminase activity was approximately 10 times lower in horse lymphoid tissue in comparison to that found in human lymphoid tissue. In horse, the highest activity was in spleen while the lowest activity was in thymus.
• 4.4. Adenosine inhibition of mitogenesis in PBL was prevented by uridine or cytidine, suggesting pyrimidine starvation as the major mechanism for adenosine toxicity.
• 5.5. Deoxyadenosine inhibition of mitogenesis in PBL was not prevented by the addition of various ribo- or deoxyribonucleosides. This and the finding that treated cells show no increase in deoxyATP suggest that some other mechanism for deoxyadenosine toxicity other than deoxyATP inhibition of ribonucleotide reductase operates in horse PBL.

     

Fetal lung metabolism: Response to maternal fasting

• 1.1. Fetal lung metabolic response to maternal fasting late in gestation was investigated.
• 2.2. Maternal fasting 4 days before term was associated with low fetal plasma glucose and insulin levels but increased levels of fetal plasma glucagon, glycerol, lactate and fatty acids.
• 3.3. Fetuses from fasted mothers showed a significant decrease in body weight (30%), lung weight (30%) and lung glycogen (46%), but no change in lung protein, phospholipid or total lung DNA, suggesting that lung size is affected more than maturation.
• 4.4. Fetal lung slices incubated in vitro showed that lactate oxidation to CO₂ equalled that of glucose in control fetal lungs and was unaffected by maternal fasting, while glucose oxidation was depressed (23%).
• 5.5. Maternal fasting significantly decreased in vitro incorporation of [U-¹⁴C]-glucose, [U-¹⁴C]lactate and [1-¹⁴C]palmitate into lung phospholipids.
• 6.6. Fetal lungs from fasted mothers showed increased conversion of lactate to glucose, indicating gluconeogenic potential by fetal lung.
• 7.7. These studies show that plasma lactate serves as an important energy fuel and substrate for lipid synthesis for the fetal lung, and maternal fasting markedly alters fetal lung metabolism.

     

Effects of a purine inhibitor,allopurinol, on urate metabolism in the german cockroach,Blattella germanica L. (Dictyoptera: Blattellidae)

• 1.1. Adult male and female cockroaches (Blattella germanica) were maintained on a positive nitrogen balance diet (66% protein) containing various levels of allopurinol (0–3%) to determine the effects of allopurinol on urate synthesis and storage.
• 2.2. Each insect was injected with [¹⁴C]hypoxanthine and after 1 week was analyzed for whole-body hypoxanthine, xanthine and urate radiolabel.
• 3.3. There was a general trend of decreased whole-body radiolabel retention, radiolabeled body urates and total-body urate content in both sexes with increasing amounts of dietary allopurinol.
• 4.4. Virgin female adults were allowed to feed on diets containing 0, 25 and 66% protein plus 0.1% allopurinol and were injected with [¹⁴C]xanthine.
• 5.5. After 1 week radiolabel content in the whole-body xanthine and urate pools was determined.
• 6.6. Females on the 0% protein diets contained less radiolabel in the whole-body and body urates than those on either 25 or 66% protein diets.

     

Effect of pH on metabolism of the glutamine carbon skeleton by renal cortical mitochondria

• 1.1. To determine the effect of altered acid-base homeostasis on the intramitochondrial metabolism of the glutamine carbon skeleton ¹⁴CO₂ production from [U-¹⁴C]glutamine by isolated rat renal cortical mitochondria was measured.
• 2.2. Mitochondria from rats with chronic metabolic acidosis either showed no change or diminished ¹⁴CO₂ production in comparison with pair fed controls.
• 3.3. By contrast, when the pH of the medium incubating mitochondria from normal rats was manipulated (pH 7.0, 7.4, 7.7), ¹⁴CO₂ production was clearly altered, but the direction and magnitude of the change depended on the glutamine concentration used (0.5 or 10.0 mM).
• 4.4. Mitochondria produced significant quantities of ¹⁴CO₂ when [1,4 ¹⁴C]succinate was used as substrate, indicating that ¹⁴CO₂ production from glutamine does not originate solely from the decarboxylation of α KG.
• 5.5. Thus chronic acidosis and pH, per se, affect intramitochondrial glutamine carbon skeleton metabolism in different fashions, but the specific mechanism cannot be elucidated using ¹⁴CO₂ production from [U-¹⁴C]glutamine.
• 6.6. Additional studies directly quantitating the metabolic products of glutamine have confirmed these findings and more precisely defined the sites of metabolic alteration.

     

Phosphoinositides and inositol phosphates in Discopyge tschudii electrocyte membranes

• 1.1. The metabolism of inositol (Ins)-containing phospholipids and inositol phosphates has been studied by following the incorporation and distribution of myo-[³ H]Ins in metabolically active electrocytes from the electric ray Discopyge tschudii.
• 2.2. The apparent initial rate of myo-[³H]Ins incorporation into total phosphoinositides was ca 8.2 fmol/mg protein/hr. Phosphatidylinositol (Ptdlns) displayed the highest levels of labelling. Lithium inhibited this incorporation probably by limiting the recycling of myo-[³H]Ins from [³H]Ins-monophosphate.
• 3.3. The formation of water-soluble products of phosphoinositides between 7 and 24 hr was 4.1 ± 0.2, 0.4 ± 0.2 and 3.0 ± 1.0 fmol/μmmol total lipid phosphorus for myo-[³H]InsP, -InsP₂ and Ins-P₃ respectively.
• 4.4. Lithium ions are shown to modulate phosphoinositide synthesis and Ins-phosphate accumulation. Ins-mono-, bis- and tris-phosphate production was enhanced 5-, 3- and 2-fold by Li +.
• 5.5. The above results suggest the participation of a C-type phospholipase and of Li-sensitive phosphatases in the modulation of phosphoinositide metabolism in the electrocyte.

     

Possible involvement of adenylylation in the modification of a 26 kDa protein in rat parotid acinar cells

• 1.1. Adenylylation, a posttranslational modification of proteins, was investigated in saponin-permeabilized acinar cells of the rat parotid gland.
• 2.2. When cells were incubated with [2,8-³H]ATP, several proteins, including a 26 kDa protein in the particulate fraction, were labeled.
• 3.3. Upon incubation of cells with [α-³²P]ATP in the presence of cAMP and 3-isobutyl-lmethylxanthine, ³²P-labeling of the 26 kDa protein was observed.
• 4.4. After treatment with snake venom phosphodiesterase, [³²P]AMP was released from the 26kDa protein. Such release was not observed when cells were labeled with [γ-³²P]ATP.
• 5.5. The ³²P-labeling pattern of proteins with [α-³²P]ATP was clearly different from that with [adenylate-³²P]NAD⁺.
• 6.6. The results suggest that the 26 kDa protein is one of the adenylylation substrates in rat parotid acinar cells.

     

Mechanism of pepsin-catalyzed aminotranspeptidation reactions

• 1.1. The tetrapeptide Ala₂-Nph₂ (where Nph = p-nitrophenylalanyl) is treated by porcine pepsin to study the mechanism of aminotranspeptidation reactions.
• 2.2. The major initial product is Ala₂-Nph and the major transpeptidation products are Nph₂ and Nph₃ accompanied by some Nph, a little Nph₄, Ala₂-Nph₃ and Ala₂-Nph₄.
• 3.3. Oligomers of Nph greater than tetramers are formed near the end of the reaction.
• 4.4. In presence of [³H]Nph, no incorporation of Nph into the transpeptidation products is observed.
• 5.5. ¹⁸O-Iabeling shows extensive incorporation of ¹⁸O atoms from [¹⁸O]water in the carbonyl oxygens of Nph residues.

     

Amino acid synthesis during hyperosmotic stress in penaeus aztecus postlarvae

• 1.1. Glycine, proline, and taurine are the quantitatively most important amino acid osmolytes in Penaeus aztecus postlarvae.
• 2.2. Taurine dominates the amino acid pool in low salinity, while proline dominates the amino acid pool at higher salinities.
• 3.3. Although not major contributors to the pool, glutamate and alanine are constitutively synthesized from [¹⁴C]glucose and [¹⁴C]glutamate under constant salinity and under hyperosmotic stress treatments.
• 4.4. Proline synthesis from [¹⁴C]-precursors is apparent under constant high (but not low) salinity and is significantly induced by hyperosmotic stress.
• 5.5. No appreciable glycine synthesis was observed from [¹⁴C]glucose or [¹⁴C]glutamate under any experimental conditions.

     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Glucosylation of mannose containing dolichyl diphosphate oligosaccharide

• 1.1. A maximum rate of dolichyl phosphate [¹⁴C]glucose synthesis from 55-day embryos was achieved at 16nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate.
• 2.2. The highest values of [¹⁴C]glucose incorporation from UDP-[¹⁴C]glucose into dolichyl phosphate [¹⁴C]glucose, dolichyl diphosphate [¹⁴C]Glc-oligosaccharides and proteins were reached at 5 min time point of incubation of liver microsomes both from embryos and sows.
• 3.3. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows.
• 4.4. The enzymatic transfer of Glc₃-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides.
• 5.5. One labelled compound was discovered in the Chcl₃-Ch₃Oh-H₂O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[¹⁴C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc₂Man₉Glc₃.