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.

     

The isolation of skeletal muscle plasma membranes from rainbow trout (Salmo gairdneri)

• 1.1. Plasma membranes were isolated from caudal flank skeletal musculature of rainbow trout by discontinuous sucrose gradient centrifugation.
• 2.2. Na⁺−K⁺-ATPase was enriched 8-fold and 5′-nucleotidase activities 4-fold in a fraction isolated at the 8–25% sucrose interface.
• 3.3. A cholesterol: phospholipid ratio of 0.37 in the plasma membrane fraction was 85% greater than that observed in adjacent subcellular fractions.
• 4.4. Electron microscopy provided morphological confirmation of enrichment and integrity of skeletal muscle plasma membranes at the 8–25% sucrose interface.

     

A comparative study on lipid peroxidation in cerebral cortex of stroke-prone spontaneously hypertensive and normotensive rats

• 1.1. Lipid peroxidation and membrane-related enzyme changes in the cerebral cortex of stroke-prone rats (SHRSP) and normotensive rats were examined at 5 and 20 weeks of age.
• 2.2. In vivo formation of thiobarbituric acid-reactant substances was higher in SHRSP at 20 weeks of age and in vitro generation of free malondialdehyde was greater in SHRSP brains, both at 5 and 20 weeks of age, as compared with those in WKY.
• 3.3. Membrane-associated enzymes such as Na/K-ATPase and 5'-nucleotidase activities were lower in 20-week-old SHRSP than in age-matched WKY.
• 4.4. These results indicate how very prone the SHRSP brain is toward lipid peroxidation and subsequent membrane-related enzyme changes.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

PLA2 activity in rat liver nuclei

• 1.1. Subcellular fractions of rat liver were assayed for PLA₂ activity.
• 2.2. The PLA₂ assay measures the release of [³ H]oleic acid from phospholipids, using labeled E. coli as substrate.
• 3.3. Nuclear fractions contained PLA₂ activity, which was Ca²⁺ dependent and could not be explained from mitochondrial, microsomal or plasma membrane contamination.
• 4.4. The V_max value of nuclear PLA₂ is 0.30 ± 0.04 pmol oleic acid/min/mg protein; its K_m value is 0.86±0.12μM, similar to that of mitochondrial PLA₂.
• 5.5. We conclude that rat liver nuclei contain PLA₂ activity.

     

Amino acid sequences around exofacial proteolytic cleavage sites of band 3 from bovine and porcine erythrocytes

• 1.1. Amino acid sequences of bovine and porcine band 3, an erythrocyte anion transporter, were determined.
• 2.2. The sequence of bovine band 3 was positioned to residues 519–599 (the numbering is based on human band 3), in which probably 6 residues were unidentified.
• 3.3. Binding site of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a potent anion transport inhibitor, was identified as Lys-539 in the bovine case.
• 4.4. A loop (residues 551–567), which provides exofacial proteolytic cleavage sites, contains only 53% homology between human and bovine, whereas the residues flanking it on either side are >84% homologous.
• 5.5. Furthermore, the loop of porcine band 3 was indicated to consist of a 6 or 7-residues short peptide as compared with those of other species.

     

Locust vitellogenin receptor: an acidic glycoprotein with N- and O-linked oligosaccharides

• 1.1. The locust vitellogenin (VTG) receptor which is embedded in oocyte plasma membranes is a glycoprotein.
• 2.2. With various lectins oligosaccharide units have been identified, among them neuraminic acid linked to Gal or GalNAc, mannose chains, Gal linked to GalNAc or GlcNAc and fucose linked to GlcNAc.
• 3.3. With specific enzymes it could be shown that mannose and most other oligosaccharides are O-linked while others like fucose are N-linked.
• 4.4. Enzymatic removal of all O-linked carbohydrates resulted in a drop of the molecular mass of the receptor protein from 200,000 to 110,000.
• 5.5. A total of N- and O-linked oligosaccharides of 54% was calculated.
• 6.6. The isoelectric point of the receptor was found to be at pH 3.4 increasing slightly after removal of neuraminic acid.
• 7.7. Removal of neuraminic acids destroyed the binding ability for VTG.

     

The characterization of the linoleic acid desaturation and elongation system in ovine placental tissue

• 1.1. The desaturation and elongation of linoleic acid has been studied in homogenates and in subfractions of ovine placental tissue.
• 2.2. The reaction was characterized in terms of pH and temperature optima, time course and protein concentration.
• 3.3. Activity was found to be confined to the 11,000g supernatant fraction of the tissue and the results suggest that the enzymes are membrane bound.
• 4.4. The cytosolic fraction and ATP were required for full activity and the reaction was inhibited by cyanide.
• 5.5. The properties of the reaction are compared with those of other desaturation systems and their implications with regard to possible reaction mechanisms are discussed.

     

Investigation of phycocyanin-membrane interaction: Promotion of membrane interactions

• 1.1. In a continuing investigation of phycocyanin-membrane surface interaction, fluorescence quenching experiments were performed with a mixture of two populations of fluorescence probe-encapsulated phospholipid bilayer vesicles in the presence and absence of phycocyanin.
• 2.2. These membrane vesicles were prepared with 1,2-dimyristoyl phosphatidylcholine (DMPC), cholesterol and a probe molecule.
• 3.3. A fluorophore was encapsulated in one population of membrane vesicles, while a quencher was encapsulated in another population of membrane vesicles.
• 4.4. The result was compared with those of experiments in the presence of other biomolecules, including albumin, cytochrome c, hemoglobin, myoglobin or RNA.
• 5.5. Interestingly, a one-third reduction of the fluorescence intensity was observed in the mixture of these two populations of membrane vesicles in phycocyanin's presence.
• 6.6. In contrast, the other biomolecules caused no significant reduction in the fluorescence intensity.
• 7.7. These findings were evidence of a phycocyanin-induced membrane perturbation.
• 8.8. This was further demonstrated by a phycocyanin-induced change in the thermotropic behavior of DMPC vesicles, as measured by differential scanning microcalorimetry.
• 9.9. Such a unique property of phycocyanin is believed to be associated with its known membrane surface-interacting character.
• 10.10. A possible phycocyanin-modulated membrane-membrane interaction was discussed.

     

Light-induced effects of several enzymes of carbohydrate metabolism in Phycomyces blakesleeanus

• 1.1. The photoregulation shown by glyceraldehyde 3-phosphate dehydrogenase and glucose 6-phosphate dehydrogenase appears to be independent of the mad gene product(s) and also independent of carotene biosynthesis regulation.
• 2.2. The photoregulation of malate dehydrogenase appeared to be dependent on the mutation of the mad and car S genes.
• 3.3. Pyruvate kinase and lactate dehydrogenase may be classified as light-independent.
• 4.4. The action of ATP and fructose 1,6-bisphosphate on the enzymes studied was generally independent of light/dark grown conditions.
• 5.5. However, the effect of fructose 1,6-bisphosphate on Phycomyces pyruvate kinase appears to be light-dependent.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

     

An improved freeze-dried blood diet for membrane feeding of glossina palpalis palpalis

• 1.1. The effect of fetal calf serum as an additive for reconstituted freeze-dried bovine and porcine blood for feeding Glossina palpalis palpalis was determined.
• 2.2. Reproductive performance of tsetse flies fed reconstituted freeze-dried bovine and porcine blood supplemented with fetal calf serum was higher than that of flies fed only freeze-dried blood.
• 3.3. Sonicated freeze-dried porcine blood supplemented with fetal calf serum resulted in a reproductive performance equivalent to that of flies fed on non-sonicated freeze-dried porcine blood and fetal calf serum.
• 4.4. A 50:50 mixture of freeze-dried porcine and freeze-dried bovine blood reconstituted and sonieated resulted in a reproductive performance equivalent to the highest ever attained for in vitro-reared G.p. palpalis.

     

Electrophoretic characterization of a hybrid between Eretmochelys imbricata and Caretta caretta (Cheloniidae)

• 1.1. An intermediate morphotype between Eretmochelys imbricata and Caretta caretta was studied in Praia do Forte, Bahia, Brazil.
• 2.2. Three enzymatic systems were successfully analyzed: SOD, lactate dehydrogenase (LDH) and esterase (EST). Isoelectric focusing of total soluble proteins of muscle and transferrin were shown.
• 3.3. Esterase exhibited nine phenotype patterns, seven in C. caretta and one in the others morphotypes. SOD phenotypes were identical in the three morphotypes. Lactate dehydrogenase and transferrins were characteristic for each species.
• 4.4. Jaccard's measure of similarity was calculated and a phenogram with the three morphotypes were constructed using isoelectric focusing of total soluble proteins.

     

Lysosomal acid deoxyribonuclease from vervet monkey livers—II: Enzymic properties

• 1.1. Primate liver lysosomal acid DNase is an endonucleolytic enzyme.
• 2.2. The enzyme has both 3'- and 5'-nucleotidohydrolase activities.
• 3.3. The oligonucleotides produced by DNase are polymers mainly about 30 mononucleotides long.
• 4.4. The Arrhenius plot shows a discontinuity with a transition temperature at 47°C, with an activation energy of 107 kJ/mol below and 67 kJ/mol above this temperature.
• 5.5. The activation enthalpy is 104kJ/mol and the entropy −0.498 kJ/mol/K.
• 6.6. The enzyme is subject to substrate inhibition and the K_m value is 159 × 10⁻³mM DNA-P.

     

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.

     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

     

Comparison of 5′-nucleotidase activities of isolated plasma membranes of two ascites cell variants

• 1.1. The glycogen-containing ascites cell line was found to have a 3–5 times higher 5′-nucleotidase specific activity than the glycogen-free variant, resulting in different substrate affinity constants of K_m = 0.14mM and K_m = 0.69mM respectively.
• 2.2. These activity differences were due to true 5′-nucleotidase as shown by its inactivation through specific inhibitors such as concanavalin A and α,β-methylene adenosine diphosphate.
• 3.3. Substrate specificity of the enzyme was similar in both cell lines, but differences were observed with respect to the pH optimum and stability.

     

Role of glutamate dehydrogenase reaction in the control of citrate pool in yeast

• 1.1. Role of NADP-glutamate dehydrogenase in the depletion of citrate was analyzed using permeabilized yeast cells.
• 2.2. Citrate was converted to 2-oxoglutarate, which was then metabolized to glutamate by NADP-glutamate dehydrogenase in the presence of ammonium ion.
• 3.3. Formation of 2-oxoglutarate plus glutamate was in good agreement with the concentration of citrate decreased. Glutamate formation can be a good indicator of the depletion of citrate, because 70% of the citrate decreased was converted to glutamate.
• 4.4. Glycolytic activity was closely correlated with the decrease in citrate under the in situ conditions.
• 5.5. NADP-glutamate dehydrogenase increased in anaerobically grown yeast cells.
• 6.6. An effective depletion of citrate by increased synthesis of NADP-glutamate dehydrogenase can explain the lowered mechanism of citrate causing glycolytic stimulation under the anaerobic growth conditions of yeast.

     

Increased muscle glucose uptake in response to chronic glyburide treatment is not related to changes in glucose transporter (GLUT4) protein

• 1.1. The mechanism of action of glyburide (a sulfonylurea) on muscle has been investigated by measuring glucose uptake and glucose transporter (GLUT4) protein levels after chronic glyburide treatment.
• 2.2. A dietary induced insulin resistant rat model (4 wk of high-fat, high-sucrose feeding) was given glyburide (2mg/kg/day) for 10 days and glucose uptake was measured in a perfused hindquarter preparation.
• 3.3. Protein levels of the GLUT4 glucose transporter were determined by Western analysis.
• 4.4. After 7 days of treatment, rats fed glyburide had lower blood glucose concentrations 2 hr (72 ± 5 vs 103 ± 12 mg/dl) and 24 hr (97 ± 7 vs 123 ± 7 mg/dl) after glyburide administration with no difference in serum insulin levels compared to vehicle treated animals.
• 5.5. Glucose uptake was approx doubled in basal state (0 insulin) in response to glyburide (2.8 + 0.4 vs 1.7 ± 0.2μ mol/g per hr).
• 6.6. Maximal insulin (100 nM) stimulated glucose uptake tended to be higher in the glyburide treated group, but did not reach statistical significance (8.0 ± 0.7 vs 7.0 ± 0.6 μmol/g per hr).
• 7.7. Western analysis revealed no significant effect of glyburide on the GLUT4 protein level in skeletal muscle.
• 8.8. These results suggest that glyburide alters glucose uptake through some mechanism other than alterations in the level of the GLUT4 glucose transporter protein.

     

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.