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.

     

Intramitochondrial reductive carboxylation of 2-oxoglutarate in adipose tissue and its contribution to fatty acid synthesis

• 1.1. The reductive carboxylation of 2-oxoglutarate was found to proceed in mitochondria of rat epididymal fat pads and rabbit perirenal adipose tissue at a rate similar to that in liver mitochondria.
• 2.2. In rat fat pads the incorporation of ¹⁴C from [5-¹⁴C]2-oxoglutarate into fatty acids via the carboxylation was suppressed by butylmalonate by 30%.
• 3.3. 2-Oxoglutarate and glutamate stimulated the incorporation into fatty acids of ¹⁴C from [2-¹⁴C]acetate in rat fat pads with the simultaneous reduction of tissue NADP. These effects persisted after inhibition of succinate dehydrogenase by malonate.
• 4.4. It is concluded that in adipose tissue 2-oxoglutarate carboxylation proceeds in both the cytoplasm and mitochondria. Therefore, it can supply carbon atoms as well as NADPH for fatty acid synthesis.

     

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.

     

Cotranslational fatty acylation of mucus glycoprotein. Addition of palmitic acid to peptidyl-tRNA occurs prior to peptide chain completion and its release

• 1.1. The fatty acylation of mucus glycoprotein nascent peptides was investigated using [³H]palmitic acid and [³⁵S]methionine-labeled peptidyl-tRNA of rat gastric mucous cells.
• 2.2. The mucus glycoprotein peptidyl-tRNA fraction was found to contain covalently bound palmitic acid in its complexes.
• 3.3. RNase digestion of the mucus glycoprotein peptidyl-tRNA released [³H]palmitic acid labeled peptides which, on SDS-polyacrylamide gel, separated into a multitude of bands ranging in size from 2000 to 60,000 Da.
• 4.4. The analyses of low molecular weight peptides revealed that palmitic acid was present in methionine-labeled peptides containing 30–43 amino acids and those of 18–25 amino acids or larger devoid of methionine, but was not identified in methionine-labeled peptides containing 10–15 amino acids.
• 5.5. The results indicate that the N-terminal fatty acylation of mucus glycoprotein nascent peptides is a cotranslational process which is occuring in an immediate vicinity of the signal peptide fragment.

     

Driving forces for the uphill transport of amino acids into epidermal brush border membrane vesicles of the sea anemone,Anemonia sulcata (Cnidaria,anthozoa)

• 1.1. The transport of amino acids into membrane vesicles prepared from epidermal tentacle tissue of the sea anemone, Anemonia sulcata, depends on an electrochemical potential difference caused, e.g. by sodium chloride gradients.
• 2.2. Potassium or choline chloride gradients energized the transport less effectively than sodium chloride gradients. Both Na⁺-ions and Cl⁻-ions were required for the amino acid transport.
• 3.3. The uphill transport of amino acids along the downhill movement of driver ions (sodium chloride gradient conditions) was characterized by an overshoot; under sodium chloride equilibrium conditions, however, an accumulation of amino acids within the vesicles could not be measured.
• 4.4. Potassium diffusion potentials in combination with valinomycin indicated that hyperpolarization (vesicle inside negative) and hypopolarization (vesicle inside positive) enhanced or depressed the accumulation of amino acids within the vesicles.
• 5.5. Being at the phylogenetic base of the Eumetazoa, cnidarians show characteristics for the transmembrane transport of amino acids comparable to those established for vertebrates.

     

Influence of dietary sources of fat on lipid synthesis in mink (Mustela vison) mammary tissue

• 1.1. The fatty acid composition of the triglyceride fraction of mink milk sampled during mid-lactation (day 28 post partum) from two nursing mink was compared to that of plasma samples and to the fatty acid composition of the feed rations used.
• 2.2. Chemical analysis of the triglyceride composition of mink milk demonstrated only minute concentrations of fatty acids with a chain length below C₁₄.
• 3.3. The saturated C_16:0- and C_18:0-unit fatty acids in mink milk made up for 24–40% of the total amount of fatty acids extracted, the remainder being represented by mono and polyunsaturated long-chain (C₁₆-C₂₄) fatty acids.
• 4.4. Preliminary in vitro experiments proved the incorporation of¹⁴C-labelled glucose, acetate or palmitate into triacylglycerols in cultures of mink mammary tissue to be linear for at least 2 hr.
• 5.5. The in vitro capacity for de novo fatty acid synthesis in mink mammary tissue using 14C-labelled glucose or acetate was low, i.e. ranging from 0.096–0.109 nmol/g (fresh tissue)/min, and amounted to only about 5% of that obtained in the case of [¹⁴C]palmitic acid incubation.
• 6.6. Following ¹⁴C-labeIled acetic or palmitic acid incubation of mink mammary tissue neither desaturation nor chain elongation was observed.
• 7.7. In response to long-term feeding on rations with two different sources of animal fat (F = fish oil or L = lard) the influence of compositional changes in dietary neutral lipids on the fatty acid composition of the lipids of mink milk is discussed.

     

Synthesis of reserved polysaccharide from wax esters accumulated as the result of anaerobic energy generation in Euglena gracilis returned from anaerobic to aerobic conditions

• 1.1. Euglena gracilis SM-ZK (a non-photosynthetic mutant), cultured in Koren-Hutner medium, containing glucose, malate and glutamate as the main nutrients, were incubated anaerobiosis for 24 hr, and then returned to aerobic conditions. Wax esters, which were synthesized from paramylon (the reserved polysaccharide) for ATP generation under anaerobiosis (wax ester fermentation) were promptly degraded immediately after the cells were replenished with sufficient O₂. A large part (about 70%) of the decomposed wax esters were converted back to paramylon.
• 2.2. When cells were fed with [1–14C]acetate or [U-¹⁴C]acetate immediately after transfer from anaerobic to aerobic conditions, radioactivity incorporated into paramylon in the cells fed with [U-¹⁴C]acetate was about 1.5-times as high as that with [1-¹⁴C]acetate, proposing that glyoxylate cycle participates in the conversion from wax esters to paramylon.
• 3.3. Paramylon synthesis from [1-¹⁴C]acetate was considerably activated by anaerobic preincubation of cells for several hours.
• 4.4. Isocitrate lyase and malate synthase occurred in cells cultured in Koren-Hutner medium, but the activities were obviously lower than those in cells grown on ethanol. These enzymes were not induced by the anaerobic preincubation.

     

The pathway of the biosynthesis of non-methylene-interrupted dienoic fatty acids in molluscs

• 1.1. The metabolic fate of 1-¹⁴C-acetate administered to the marine bivalve mollusc Mytilus edulis was investigated.
• 2.2. The active incorporation of the label in 20:2 non-methylene-interrupted dienoic (NMID) fatty acids was found.
• 3.3. Acetate incorporation patterns and specific radioactivity of mussel acids suggest that 22:2Δ7,13 and 22:2/gD7,15 arose by C₂ elongation of 20:2Δ5,11 and 20:2Δ5,13 respectively.
• 4.4. The proposed pathway of NMID fatty acid biosynthesis in molluscs is discussed.

     

Fate of ingested leucine and glucose in the sea star,Asterias rubens,during its annual reproductive cycle

• 1.1. The distribution of radiolabel from L-leucine [¹⁴C-UL] and D-glucose [¹⁴C-UL] was measured in the sea star Asterias rubens at 1, 6 and 24 hr after oral administration.
• 2.2. Incorporation of the label from both compounds was observed in pyloric caeca, coelomocytes and ovaries even after an incubation time of 1 hr.
• 3.3. Highest incorporation from both precursors was found in proteins, while substantial radioactivity was present in the amino acids, organic acids and neutral components. Lipids were hardly labelled from leucine and only slightly from glucose.
• 4.4. Radioactivity in proteins and lipids increased with increasing incubation time. No significant differences were found in the distribution patterns of radiolabel during the reproductive cycle.
• 5.5. The data obtained are discussed in terms of current knowledge on the translocation of nutrients in echinoderms.

     

Metabolic utilization of leucine in a fat and a lean line of chicken

• 1.1. In vivo incorporation into body lipids and breast muscle proteins from l-[U-¹⁴C]leucine was studied in genetically lean or fat male chickens, fed or starved, 1 or 24 hr after intraperitoneal injection.
• 2.2. Lipogensis and portein synthesis from labelled leucine were significantly higher in fat chickens than in lean birds, particularly in those in the fed state.
• 3.3. Radioactivity in the free amino acid pool was greater in fat birds irrespective of the nutritional state.
• 4.4. However, utilization of injected l-[U-¹⁴C]leucine for lipogenesis was no more than 2%.

     

Generation of C3- and C2-deuterated l-lactic acid by human erythrocytes exposed to d-[1-13C]glucose,d-[2-13c]glucose and d-[6-13C]glucose in the presence of D2O

• 1.1. The generation of C₂- and C₃-deuterated l-lactate was monitored by ¹³C NMR in human erythrocytes exposed to d-[1-¹³glucose, d-[2-¹³C]glucose or d-te-¹³C]glucose and incubated in a medium prepared in D₂O.
• 2.2. The results suggested that the deuteration of the C₁ of d-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C₁ of d-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C₃ of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D₂O.
• 3.3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C₃ during interconversion of the 2-keto acid and l-alanine in the reaction catalyzed by glutamate-pyruvate transaminase.
• 4.4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-¹³C]pyruvate.
• 5.5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates.

     

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.

     

The influence of calcium and magnesium on sea bass (Dicentrarchus labrax) intestinal brush border membrane purification and activity

• 1.1. The activity of brush border enzymes (alkaline phosphatase, maltase, sucrase, trehalase, leucine amino peptidase) was higher in purified membranes prepared with calcium. The contamination of these membranes with basolateral membranes was also lower (1.27 for Na-K-ATPase activity ratio).
• 2.2. The extraction of brush border lipids was carried out according to Folch adapted method. Two dimensional thin layer chromatography was used to separate the phospholipidic fractions. Fatty acids of phospholipids were analysed using gas chromatography after acid transmethylation (column SP 2330).
• 3.3. Phospholipids are composed of phosphatidylcholine (PC: 33%), phosphatidylethanolamine (PE: 30%), sphingomyeline (SM: 21%), phosphatidylserine (PS: 14%) and phosphatidylinositol (PI: 2%). 4. PC, PE and PS are characterized by high levels of unsaturated fatty acids (monounsaturated MUFA: 21.5% and polyunsaturated PUFA: 34.9%). The most abundant PUFA belong to the (n-3) family [18:3 (n-3), 20:5 (n-3) and 22:6 (n-3)].
• 4.5. Fatty acids from sphingomyelin of purified membranes have low proportions of PUFA (13.5%) but higher proportions of MUFA (39.5%).
• 5.6. No specific differences were found between calcium and magnesium prepared membranes.
• 6.7. The low content in LPC and the absence of LPE confirmed the absence of major structural lipids transformation during the membrane purification with calcium or magnesium.
• 7.8. Glycine transport was measured during 10 sec at different temperatures using the rapid filtration technique. Glycine transport was higher with Na⁺ than with K⁺. In the presence of Na⁺, this transport increases with temperature.
• 8.9. Arrhenius curves were mono phasic without obvious breakpoint and indicated no phase transition in the lipid bilayer.
• 9.10. A significant Na⁺ dependent glycine transport has been characterized at low temperatures (0°C) which suggests a possible role of membrane polyunsaturated fatty acids in the control of glycine transport.

     

Chemical feeding stimulants for the herbivorous fish,Tilapia zillii

• 1.1. Chemical feeding stimulants for an herbivorous fish, Tilapia zillii have been determined by fractionation and bioassay of substances derived from a model food plant.
• 2.2. Stimulation was produced by amino acids; glutamic acid, aspartic acid, serine, lysine and alanine produced the bulk of stimulatory activity.
• 3.3. These amino acids are among the most abundant in the test plant, and are markedly different from the amino acids found to stimulate feeding in carnivorous fish.
• 4.4. On the basis of these results, a chemically-mediated mechanism of feeding niche separation is postulated.

     

A comparative study of molluscan and crustacean chitin proteoglycans by carbon-13 NMR spectroscopy. Identification of carbohydrate and amino acid contributions and the determination of amino acid chemical shifts in anhydrous formic acid

• 1.1. ¹³C-NMR spectra of formic acid solutions of chitin proteoglycans from cephalopod pen, lamellibranch siphon sheath and crab cuticle have been determined.
• 2.2. Carbohydrate and amino acid components provide well-defined resonances, completely assignable in the case of hexosamine and partially so for protein amino acids.
• 3.3. The individually unique spectra contain information of compositional and chain environment nature.
• 4.4. Spectral data for each protein amino acid, as a formic acid solution, is presented and compared with values for chemical shifts of amino acids and peptides in water.

     

In vitro absorption of [14C]leucine during inflammation and the effect of antiinflammatory drugs in the jejunum of rats

• 1.1. Inflammation was induced in the hindlegs of rats by formalin injection and the in vitro absorption of [¹⁴C]leucine was studied.
• 2.2. Treatment of rats with formalin caused a reduction in the in vitro absorption of leucine from the mucosa of jejunum.
• 3.3. Oral administration of oxyphenbutazone or a herbal anti-inflammatory drug (Withania somnifera) prior to formalin injection resulted in no alteration in the jejunal absorption of [¹⁴C]leucine.

     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus)

• 1.1. The in vitro metabolism of [³H]benzo[a]pyrene (BP) and [¹⁴C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo.
• 2.2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [−] enantiomer) and smaller amounts of BP-4,5-diol.
• 3.3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides.
• 4.4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates.
• 5.5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25–30 days. The major adduct was anti-BPDE-deoxyguanosine.

     

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.

     

Isolation and partial amino acid sequence of A 78 kDa porcine gastrin-binding protein

• 1.1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle¹⁵]-gastrin; 17.
• 2.2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides.
• 3.3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55–61). The remaining 7 peptides were derived from the gb-subunit of the gastric H⁺/K⁺-ATPase.
• 4.4. The gastrin-binding activity remained in association with p78, and could be separated from the P-subunit of the gastric H⁺K⁺-ATPase, during chromatography on tomato lectin-Sepharose.
• 5.5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.