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.

     

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.

     

Ependymins and their potential role in neuroplasticity and regeneration: Calcium-binding meningeal glycoproteins of the cerebrospinal fluid and extracellular matrix

• 1.1. Ependymins are unique, highly divergent secretory proteins of the fish endomeninx. Thus far, no homologous sequences have been characterized in mammals.
• 2.2. Soluble ependymins are the predominant constituents of the cerebrospinal fluid of many teleost fish. A bound form of these glycoproteins is associated with the extracellular matrix probably with collagen fibrils. The latter may be the functional form of ependymins.
• 3.3. Ependymins bind Ca²⁺ via N-linked sialic acid residues leading to a conformational transition.
• 4.4. The molecular function of ependymins seems to be related to cell contact phenomena involving the extracellular matrix. For example, adhesive or anti-adhesive interactions may possibly influence ingrowing axons.

     

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.

     

Preparation and characterization of biotinylated probes for the β-interferon receptor

• 1.1. Recently we described the isolation of the β-interferon receptor [Zhang et al. (1986) J. biol. Chem. 261, 8017–8021]. A highly purified product was obtained but in low quantities.
• 2.2. The use ofbiotinylated β-interferon as a ligand represents an alternate approach to receptor isolation.
• 3.3. We have prepared and characterized the derivatives N-(biotinyl)- and N-(biotinyl-ϵ-aminocaproyl)-recombinant human [Ser¹⁷-interferon β (B- and BC-recHulFNβ).
• 4.4. Biotin incorporation does not result in any loss of antiviral activity, demonstrating the recognition of the derivative by the cell receptor.
• 5.5. The biotinylated recHuIFNβ binds specifically and reversibly to succinoylavidin or guanidine thiocyanate-stripped succinoylavidin linked to a Sepharose matrix.
• 6.6. Comparison of the competition curves obtained with [¹⁴C]biotin and [³H]biotinyl recHuIFN, in the presence of increasing concentrations of biotin suggests that the IFN moiety of the derivative has little effect on the affinity of biotin for avidin.
• 7.7. Biotinylated recHuIFNβ derivatives represent useful probes for the β-IFN receptor.

     

Pest sequences in nuclear proteins

• 1.1. Most of proteins which are rapidly degraded inside eukaryotic cells have been found to contain amino acid sequences (PEST sequences) enriched in proline, acidic residues (glutamic acid and/or aspartic acid) and hydrophilic residues (serine and threonine) (Rogers et al. (1986) Science234, 364–368).
• 2.2. This correlation was tested on nuclear proteins and a close relationship was found between nuclear protein stability and the presence of PEST regions.
• 3.3. Nuclear proteins with structural functions which can be considered as stable components of cell nuclei generally lack PEST sequences.
• 4.4. In contrast, regulatory nuclear factors which have specific and transient functions generally possess at least one PEST sequence.

     

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.

     

Small membrane-associated gtp-binding proteins of catecholamine-secreting cells

• 1.1. Four GTP-binding proteins (23–27 kDa) were identified in membranes from PC12 cells by [α³²P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels.
• 2.2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K⁺-depolarization or even after addition of C²⁺to digitonin-permeabilized cells.
• 3.3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca²⁺.
• 4.4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity.
• 5.5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane.
• 6.6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

     

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%.

     

Absorption and blood clearance of labelled carotenoids ([14C]astaxanthin, [3H]Canthaxanthin and [3H]zeaxanthin) in mature female rainbow trout (Oncorhynchus mykiss)

• 1.1. Using one force-fed meal, eight mature female rainbow trout received [¹⁴C]astaxanthin ([¹⁴C]Ax) with [³H]canthaxanthin ([³H]Cx; N = 3) or with [³H]zeaxanthin ([³H]Zx; N = 5).
• 2.2. Approximately 200 μl of blood were collected via caudal puncture every 24 hr for 4 days. After 96 hr, the fish were killed and pyloric caeca (P.C.) from the duodenal intestine (D.I.) section, ileal intestine (I.I.), and posterior intestine (P.I.) were dissected out.
• 3.3. In the blood, Ax levels were higher than Cx followed by Zx levels.
• 4.4. This corresponds to their respective absorption by the trout as was confirmed by their relative concentrations in P.C., I.I. and P.I.
• 5.5. However, blood clearance was similar for all three compounds. [¹⁴C]Phoenicoxanthin ([¹⁴C]Px) was detected as a reduced metabolite of [¹⁴C]Ax in all gut sections.

     

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.

     

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.

     

A Robust and Versatile Automated Glycoanalytical Technology for Serum Antibodies and Acute Phase Proteins: Ovarian Cancer Case Study

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Highlights

• •Quantitative high-throughput glycoanalytical technology as a diagnostic tool for ovarian cancer detection.
• •Multiplexed approach harnessing N-glycan data for six glycoproteins from a single biological sample.
• •Detailed characterization of human serum N-glycans from antibodies IgG, IgM and IgA and acute phase proteins transferrin, haptoglobin and alpha-1-antitrypsin.
• •Structural differences in antibody and acute phase protein glycosylation for mechanistic insights.

     

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.

     

Bovine aorta contains at least two related forms of heparan sulphate proteoglycan

• 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
• 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
• 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
• 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
• 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [³⁵S]sulphate and [³H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
• 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
• 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.

     

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.

     

A study of the effect of heat shock and metal ions on protein synthesis in Neurospora crassa cells

• 1.1. Neurospora cells were grown at 28°C for 14hr and subjected to heat shock (HS) at 48°C for 45 min. Protein synthesis profiles, monitored by labelling with [³⁵S]methionine and one and two-dimensional electrophoresis, revealed nine heat shock proteins (HSPs).
• 2.2. Crossed-immunoelectrophoresis revealed five polypeptides in the shocked cell extracts that were not detectable in normal cells.
• 3.3. Synthesis of HSPs occurred rapidly during the shock treatment and ceased upon transfer to normal conditions. One of the HSPs—~43 K in size—may be a developmentally-regulated protein.
• 4.4. Metal ions—cadmium, zinc, manganese, copper—did not elicit a stress response when used alone but appeared to modulate the heat shock response.

     

Localization of the glucocorticoid receptor in rat liver cells: Evidence for plasma membrane bound receptor

• 1.1. The cytoplasmic glucocorticoid receptor of rat liver cells is in part recovered in the plasma membrane fraction.
• 2.2. After in vivo administration of [³H]dexamethasone, 0.35% of the radioactivity recovered is bound on plasma membranes.
• 3.3. Dexamethasone also binds in vitro specifically to plasma membranes. Expressed as fmol/mg protein, binding of dexamethasone to plasma membranes is comparable to binding to the soluble cytoplasmic fraction (cytosol).
• 4.4. Using polyclonal antibody to the glucocorticoid receptor and the indirect immunofluorescence technic, an intense decoration of the plasma membranes is observed, denoting a high concentration of glucocorticoid receptor on plasma membranes.
• 5.5. The localization of the receptor on plasma membranes could be of potential importance for its interaction with agents (mitogens, growth factors) initially acting on the cell membrane, regulating subsequent cell proliferation and growth at the level of the cell nucleus.

     

Modulation of gastric mucosal calcium channel activity by mucus glycoprotein

• 1.1. The effect of gastric mucus glycoprotein on the activity of calcium channel isolated from gastric epithelial cell membrane was investigated. The ⁴⁵Ca²⁺ uptake into the vesicle-reconstituted channels, while only moderately (14%) affected by the intact mucus glycoprotein, was found significantly inhibited (59%) by the acidic glycoprotein fraction. This effect was associated with the sialic acid and sulfate ester groups of the glycoprotein, as their removal caused a loss in the inhibition.
• 2.2. The channel complex in the presence of epidermal growth factor (EGF) and ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels showed a 50% increase in ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 3.3. The channel protein phosphorylation was inhibited by the acidic mucus glycoprotein, which also interfered with the binding of EGF to the channel protein. The inhibitory effect was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoprotein inhibitory capacity following their removal.
• 4.4. The results suggest that the acidic gastric mucus glycoproteins, by modulating the EGF-controlled calcium channel phosphorylation, play a major role in gastric mucosal calcium homeostasis.

     

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.