Effect of lindane on phosphatidylinositol synthesis by cerebral cortex after acute and subchronic treatment

• 1.1. The incorporation of myo-[2-³H]inositol into phosphatidylinositols was unmodified in brain cortex miniprisms from convulsant rats.
• 2.2. However, the incorporation had increased by 300–400% in non convulsant rats which had received the same amount of lindane at a lower concentration.
• 3.3. This result suggests that phosphatidylinositols are implicated in the convulsion syndrome.
• 4.4. Experiments with lindane added in vitro were performed with both subchronically lindane intoxicated and untreated rats.
• 5.5. The results show an interesting lack of parallelism.
• 6.6. This might indicate the development of some resistance to the effects of lindane, possibly as the result of complex compensatory changes in inositol lipid biosynthesis.

     

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.

     

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.

     

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.

     

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.

     

Calcium alters the properties of [3H]diazepam binding sites in rat brain membranes

• 1.1. [³H]diazepam ([³H]DZ) was used as a ligand to study the effects of Ca²⁺ on benzodiazepine binding to rat brain membranes.
• 2.2. [³H]DZ bound at a single class of binding sites showing K_D and B_max values of 5.4 nM and 852 fmol/mg protein respectively. These values are consistent with previous reports.
• 3.3. Amongst the various divalent cations tested Mg²⁺, Fe²⁺, Cd²⁺, and Sr²⁺ had no significant effect on [³H]DZ binding. Mn²⁺, Ba²⁺, Co²⁺, Ni²⁺ and La²⁺ enhanced radioligand binding, whereas Ca²⁺, Zn²⁺ and Pb²⁺ inhibited [³H]DZ binding to brain freeze-thawed membranes.
• 4.4. The inhibition of [³H]DZ binding by Ca²⁺ was concentration-dependent. 50% inhibition occurred at a Ca²⁺ concentration of 5.6mM. The Hill coefficient for the inhibition was 1.03, displaying noncoperativity. The effect of Ca²⁺ on [³H]DZ binding could be prevented by La³⁺ but was not reversed by EGTA.
• 5.5. A kinetic analysis of Ca²⁺ inhibition of [³H]DZ binding indicates that Ca²⁺ inhibited competitively. Analysis of binding isotherms indicates that both K_D and B_max were altered at the [³H]DZ binding sites. The marked increase in K_D value in the presence of Ca²⁺ (5 mM) can be explained by a drastic increase in the dissociation rate constant.
• 6.6. It was suggested that Ca²⁺ may induce a conformational change in the diazepam binding sites on rat brain membranes. The unchanged Hill coefficient in the presence or absence of Ca²⁺ indicates that a single population of binding sites was labeled by [³H]DZ.
• 7.7. The calmodulin antagonists, trifluoperazine and W-7 were weak inhibitors of [³h]dz binding.

     

Selective effect of melatonin on the proliferation of lymphoid cells

• 1.1. The present study was designed to investigate the effect of melatonin on the proliferation of normal lymphocytes and certain T-lymphomas and myelomas under in vitro conditions.
• 2.2. The results revealed that administration of 200 μM melatonin inhibited significantly the incorporation of [³H]thymidine into both normal mouse and human lymphocytes and T-lymphoblastoid cell lines.
• 3.3. On the contrary, melatonin provoked an increase of myeloma cell proliferation.
• 4.4. The influence of melatonin on hybridoma cell lines was negligible.
• 5.5. Collectively, these data demonstrated that the chief pineal indole affect selectively the processes of lymphoblastoid cell growth.

     

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.

     

Glucose metabolism in a kangaroo (Macropus robustus erubescens) and a similar size eutherian herbivore,the feral goat

• 1.1. Glucose pool size, space, entry rate, and turnover time were estimated from the specific radioactivity vs time curves of [³H] and [¹⁴C]glucose administered as a single injection in the euro (Macropus robustus erubescens) and the sympatric feral goat (Capra hircus).
• 2.2. Digestible energy intake was greater (P < 0.05 ± SE) in the goat than in the euro (798 ± 64 vs 624 ± 31kJ/kg^0.75 × day).
• 3.3. However, there were no significant differences between the two species in parameters of glucose metabolism.
• 4.4. The use of an implantable osmotic infusion pump to deliver isotopic glucose showed promise as a means of avoiding the stress involved with the single injection technique.

     

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.

     

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.

     

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.

     

Gluconeogenesis in hepatocytes determined with [2-13C] acetate and quantitative 13C NMR spectroscopy

• 1.1. In the present study the major metabolic pathways of glucose metabolism were determined in isolated liver cells using [2-¹³C]acetate and ¹³C magnetic resonance spectroscopy.
• 2.2. The relative reaction rates of glucose synthesis to the TCA cycle were determined from the ¹³C distribution in glucose where the overall ¹³C enrichment of glucose was 6.41 ± 1.94% (mean ± SD; n = 6) and the mean ¹³C enrichment of C1, C2, C5, C6 to C3, C4 was 2.63 ± 0.30.
• 3.3. Since the distribution of tracer in glucose is a function of the relative entry rates of pyruvate to acetyl-CoA into the oxaloacetate pool this was calculated to be 0.32 ± 0.15 and the factor for carbon exchange (1/P) between the gluconeogenic pathway and the TCA cycle was calculated to be 1.03 ± 0.20.
• 4.4. With this carbon exchange factor and the approximated ¹³C enrichment of acetyl-CoA the intramitochondrial ¹³C enrichment of phosphoenolpyruvate was calculated and the “true” rate of hepatic gluconeogenesis from phosphoenolpyruvate estimated.
• 5.5. Since acetate was metabolized solely in liver cells the ¹³C enrichment of acetyl-CoA could be approximated from that of 3-hydroxybutyrate.
• 6.6. The carbon 13 enrichment of 3-hydroxybutyrate and phosphoenolpyruvate was 5.89 ± 0.90% and 5.96 ± 1.67%, respectively.
• 7.7. The per cent gluconeogenesis from phosphoenolpyruvate calculated as the ratio of the ¹³C enrichment of glucose to that of 3-hydroxybutyrate times 1/P was 107 ± 8%.
• 8.8. In this study the validity of assessing isotopic exchange at oxaloacetate as suggested by Katz [Katz J. (1985) Am. J. Physiol.248, R391–R399] when interpretation of the data are not obscured by pseudoketogenesis.
• 9.9. Magnetic resonance spectroscopy provides direct information about intramolecular tracer distribution by which flux rates in major metabolic pathways are derived.

     

Effects of transforming growth factor-β on collagen gene expression and collagen synthesis level in mineralizing cultures of osteoblast-like cell line,MC3T3-E1

• 1.1. High levels of type I collagen mRNA and [³H]proline incorporation into collagenase digestable protein by MC3T3-E1 cells were detected during the first 7 days of culture, after which they declined.
• 2.2. Type I collagen gene expression was stimulated by TGF-β in the early culture stage when the collagen gene expression was fully functioning.
• 3.3. However, these stimulatory effects disappeared at the differentiation stages. Although collagen gene expression was stimulated by TGF-β (2.0 ng/ml) in early culture, collagen synthesis in medium was not.
• 4.4. This study shows that collagen synthesis and collagen gene expression were affected by the state of differentiation in MC3T3-E1 cells and that the rate of stimulation by TGF-β in collagen gene expression decreased over time in culture.

     

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 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.

     

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%.

     

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.

     

Demonstration of highly specific and sensitive antibodies to a naturally occurring tetrahydroisoquinoline alkaloid,salsolidine

• 1.1. We report for the first time on the production and characterization of antibodies against a naturally occurring tetrahydroisoquinoline, namely salsolidine (6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline).
• 2.2. Immunogen synthesis was carried out by coupling the hapten salsolidine to bovine serum albumin (BSA) as carrier protein on the basis of reductive amination.
• 3.3. By immunization of rabbits with salsolidine-BSA conjugate antisalsolidine antibodies were produced.
• 4.4. At a final dilution of 1:1700 the highest-litre antiserum bound 35% of 0.21 pmol [³H] salsolidine. This antiserum was used to develop a radioimmunoassay for salsolidine.
• 5.5. Cross-reactivity studies revealed a high specificity of the antiserum to the hapten.
• 6.6. The antibodies had a high affinity to salsolidine (K_a = 1.5 × 10⁹ M⁻¹).
• 7.7. Standard curves covered a measuring range of 0.5–70 pmol/tube and the detection limit was found to be 0.27 pmol/tube.